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COPYRIGHT DEPOSIT. 



A LABORATORY 
GUIDE IN HISTOLOGY 



By 
CHARLES H. DeWITT, B. S. 

INSTRUCTOR IN ANATOMY IN THE MEDICAL 

DEPARTMENT OF VALPARAISO 

COLLEGE. 



VALPARAISO, INDIANA 

CHARLES H. DeWITT 

I9O4 



&k^ 5 



LIBRARY of CONGRESS 
Two Copies Received 

NOV 28 1904 

Copyngnt tniry 
CLASS O^ XX& Noj 

/ ccZc ■; 

COPY B. 



COPYRIGHT I904 

BY CHARI.ES H. DeWITT 

Valparaiso, Ind. 



Printed and Bound by 
Brock & Rankin, Chicago 



PREFACE 



The author has felt the need of a laboratory guide in this subject which 
would meet his own particular requirements and that is the only excuse offered 
for this work 

It has been the aim of the author to give the student such help as experience 
has shown that he needs, and to direct his study by question rather than by 
direct statement. 

This book has been designed to supplement the text and it presupposes 
that the student has had lectures upon the topics, as well as his ,own text-book 
study, and it should be used in no other way. 

Experience has shown that while the text-books have excellent illustrations 
the student does not use them as he should in his microscopic study of the 
tissues. With this in view this "work has been illustrated by sketches and 
photo-micrographs. These have been made by the author from his own pre- 
parations or those of his students, unless otherwise credited. The aim has 
not been to represent details of structure so much as it has been to show the 
structures as the student finds them in his own specimens. The camera lucida 
was used for most of the work, and the structures have been carefully labeled 
to aid the student in their identification and study. 

It will perhaps be said that the student has received too much help and that 
his drawings will be made from those in his laboratory guide rather than from 
the section under his microscope. While this may be true to some extent, yet 
the fact that he has a definite plan for the study of each section, and that he 
knows just how to proceed, outweighs this objection in the opinion of the 
author who has never known a beginner in the study of Histology to say that 
he has had too much light thrown upon the subject. 

To a certain extent histological technique has been combined with labora- 
tory directions for study since the student proceeds more intelligently in this 
way. While it may seem unnecessary to repeat the directions for staining and 
mounting each section, yet students are found in every class to w T hom such 
advice is always helpful. 

Drawings and sketches should be made even though the average medical 
student considers it a waste of time, for only in this way will the different 
structures and structural relations be fixed in mind. Sketching and drawing 
are very necessary adjuncts to thorough anatomical study either macroscopic 
or microscopic. 

Only those methods in common use by students are described, detailed ex- 
planations and special technique being omitted since there are plenty of splendid 
works devoted to these things. The whole aim has been to give the student a 
simple and practical working guide. Corrections and suggestions from teachers 
of Histology will be greatly appreciated. 

The author is indebted to his fellow-teachers in Valparaiso College for 
many helpful suggestions and criticisms which have aided very greatly in the 
preparation of this work. CHARLES H. DeWITT. 

Valparaiso, Indiana, November i, 1904. 




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DRAWING OF A BAUSCH AND LOMB MICROSCOPE— Yingling. 



General Suggestions to the Student- 
After the student has been assigned a place at the table and a 
locker he will be held strictly to account for the proper care of the 
same. 

Each student will be assigned a microscope and all of the 
necessary material upon the presentation of his laboratory card 
or breakage ticket. He will be carefully instructed in the use and 
care of the microscope and will be expected to use and care for the 
same in the proper manner. Any injury to the microscope due to 
carelessness upon the part of the student must be made good by the 
student causing such injury. If the microscope does not work 
properly, or if it needs an} r attention, report it to the instructor 
at once. 

Reagents of any kind must not come in contact with any part 
of the microscope. Clean and dry the slide before placing it upon 
the stage of the microscope. 

Filter paper, blotters, and waste material should be placed in 
the jars provided for that purpose. Such material must not be 
thrown upon the floor, table, or in the locker. 

The stains, oils, and alcohols most commonly used will be 
furnished to the student in proper receptacles. Special stains and 
reagents will be secured from the instructor and should be returned 
to him at the close of the laboratory period. The stains and other 
reagents should be protected from dust and evaporation when not 
in use. 

The drawings and sketches suggested must be made from the 
specimen while in the laboratory. They should be made with a 
hard pencil without much shading. A judicious use of colors is 
helpful. Drawings and their parts must be neatly labeled. The 
magnification should be indicated wherever possible. 

Formulae for stains and reagents will be found at the close of 
this work. The student is earnestly desired to familiarize himself 
with them so that his work may be performed with a greater 
degree of intelligence. The student is encouraged to ask ques- 
tions bearing upon his work at all times. 

5 



6 A LABORATORY GUIDE IN HISTOLOGY. 

General Explanation of the Process of Tissue Preparation. 

(1) Killing and Fixing. The fresh tissue must be treated 
with a killing and fixing agent in order that the cells may be killed 
before undergoing post-mortem changes. They must now be fixed 
in that condition and the cell contents rendered insoluble to the 
reagents used in the further treatment of the tissue. The pieces 
of tissue should not be large so that the killing and fixing may be 
the more readily accomplished. The volume of the reagent should 
be from fifteen to fifty times that of the material. 

(2) Washing. With the exception of the alcohols and formalin, 
most fixing agents must be washed out as completely as possible 
before proceeding further, for the reason that some fixing agents 
will hinder the action of the stains, if present in the tissue in excess, 
and in other cases precipitates form which must be removed. The 
washing is usually done in running water, but after certain killing 
and fixing fluids, alcohol must be used. If running water is not 
accessible a large volume of water should be used and it must be 
changed frequently. The best results are obtained by covering 
the vessel containing the tissues with mosquito netting to prevent 
the loss of the tissue and placing it under the tap so that the water 
may drip into the vessel constantly. 

(3) Hardening with Alcohol. After washing in water the 
tissue will require further hardening and dehydration for which 
alcohol is usually used. This must be a gradual process, therefore, 
after washing in water place the tissue in 35% alcohol for several 
hours. Then use 50-60% alcohol for five or six hours, and store the 
tissue in 80% alcohol if not wanted for immediate use. If it is 
desired to imbed the tissue at once tranfer the tissue from the 80% 
alcohol to 95% where it is left for several hours, depending upon 
the size of the pieces and the character of the tissue. Transfer to 
absolute alcohol to complete the dehydration. This process is 
very important since a very slight amount of water in the tissue will 
ruin it, or at least impair its value for histological study. 

(4) Clearing. Up to this point the process is the same whether 
the tissue is to be imbedded in celloidin or paraffin. Xylol or chloro- 
form are the clearing agents commonly used prior to imbedding in 
paraffin and ether for imbedding in celloidin. The use of a clearing 



A LABORATORY GUIDE IN HISTOLOGY. 7 

agent at this point is to remove the alcohol and render the tissue 
transparent, removal of the alcohol being absolutely essential. The 
transfer from the absolute alcohol to the clearing oil should be made 
gradually. 

(5) Imbedding. For some purposes it is merely necessary to 
surround the tissue with the imbedding substance, but for most 
work the tissues must be penetrated by and completely saturated 
with the imbedding medium. This latter is known as interstitial 
imbedding. The choice of imbedding material depends upon the 
use to which the tissue is to be put, and the thickness of the 
sections desired. The transfer from the clearing oil to the medium 
used for imbedding should be a gradual process. A general or 
suggestive outline for imbedding in celloidin and paraffin will be 
found below. 

(a) Celloidin Imbedding. 

1 . Transfer from absolute alcohol to equal parts of ether 
and absolute alcohol for 6 to 12 hours. Why ? 

2. Transfer to pure ether for 6 to 12 hours. Wh)^? 

3. Place the tissue in thin celloidin for 12 to 48 hours. 

4. Place the tissue in thick celloidin for 12 to 48 hours. 

5. Fasten to Blocks. Select from the jar of alcohol in 
which they are kept a pine block having an end surface slightly 
larger than the piece of tissue to be blocked. Dip the end of 
the block into ether-alcohol and after a moment into the thick 
celloidin. Remove the piece of tissue from the thick celloidin, 
place in the desired position upon the end of the block and 
press it somewhat firmly against the block. Let it dry a 
moment and dip it into the thick celloidin or pour a little of the 
celloidin over the surface so as to form a coat of celloidin 
around the tissue. After drying a moment in the the air place 
the block in chloroform for a few minutes to harden the 
celloidin, and then complete the hardening by placing the block 
in 80% alcohol. 

6. Cutting Sections. The sections should be cut with a 
sliding microtome. The tissue and knife should be flooded 
with 80% alcohol while cutting, and as soon as cut the sections 
should be transferred with a camel' s-hair brush from the knife 
to 80% alcohol where they remain until desired for staining. 



8 A LABORATORY GUIDE IN HISTOLOGY 



7. Staining and Mounting. Hematoxylin and Eosin 

Method. 

1. Transfer the sections from the 80% alcohol to 

water where they are to remain for one or two minutes. 

2. Stain in hematoxylin, 1 to 5 minutes. 

3. Wash in tap- water, 1 minute. If the section is 
overstained place it for a few seconds in acid-alcohol and 
wash thoroughly in water. Instead of acid-alcohol a 2-3% 
solution of ammonia alum may be used. The section may 
remain in this solution 5 to 10 minutes after which wash 
in water. 

4. Stain in eosin, 10 seconds to 1 minute. 

5. Dehydrate in alcohol. 35%, 50%, 80%, 95%, 30 
seconds to 1 minute in each up to the 95% alcohol in which 
it should remain for 2 or 3 minutes. 

6. Clear in Eycleshymer's mixture, 10 to 20 minutes. 

7. Remove the excess of the oil with a blotter, add 
a drop of Canada balsam, and cover with a clean, dry, cover- 
glass. The cover glass should be held in the cover-glass 
forceps, started at one side of the section, and lowered 
gradually so as to drive out the air. 

8. Label. The label should be numbered, the name and 
direction of the section given, and the name or initials of the 
student should be placed in the lower right hand corner. 

9. General Remarks. The above is meant to be sugges- 
tive merely and it must be varied according to circumstances. 
The student will soon learn by his own experience as to the 
time required for the various operations. Should the section 
become cloudy after adding clearing oil it is evidence that the 
dehydration has not been complete and the section should be 
placed in 95% alcohol until complete^ dehydrated. If the 
section is cloudy after adding balsam, and it did not cloud 
while in the oil, it shows that the clearing was not complete, 
for the oil must not only render the section transparent, but it 
must remove the alcohol which is not a good solvent for the 
balsam. 

(b) Paraffin Imbedding. 

1. Transfer from absolute alcohol to equal parts of 
absolute alcohol and chloroform, 6 to 12 hours. 



A LABORATORY GUIDE IN HISTOLOGY. 9 



2. Pure chloroform, 6 to 12 hours. 

3. Chloroform saturated with paraffin (cold) , 5 to 6 hours. 

4. Chloroform saturated with paraffin fin paraffin oven), 
1 to 2 hours. 

5. Soft paraffin in paraffin oven, 1 to 3 hours. 

6. Hard paraffin, 5o°-54°, 1 to 2 hours. 

7. Imbed in paper boxes. Using paper of firm texture 
make small boxes by molding the paper about wooden blocks 
of suitable size. The instructor will show you how this is 
done. Number the box to correspond with the number in 
your tissue list. Place the tissue in the box with the surface 
from which it is desired to cut sections resting on the bottom 
and cover with the melted paraffin. Cool quickly by holding 
the box in ice-water, or in cold running water. After the 
paraffin is cold remove the paper and mark the paraffin block 
with a number to correspond with that of your tissue-record. 

8. Cut sections. 

9. Fasten the section to a clea?i slide with Mayer's fixative. 

10. Heat carefully to melt the paraffin and place it in 
turpentine to remove the paraffin, after which treat it with 
xylol, 1 to 2 minutes. 

11. Treat the slide with absolute alcohol, 1 to 3 minutes; 
95% alcohol, 1-2 minutes ; 80% alcohol, 1 minute ; 50% alcohol, 
1 minute ; water, 1 to 3 minutes. 

12. Stain in hematoxylin, 1 to 5 minutes. If overstained 
treat as for celloidin sections. The staining is more precise if 
overstained and then differentiated with acid-alcohol or am- 
monia alum. Wash thoroughly after either acid-alcohol or 
ammonia alum. 

13. Stain in eosin, and dehydrate as for celloidin sections. 

14. Clear with oil of cloves, 5 to 15 minutes, mount in 
balsam, and label. 

The student should consult his text-book in Histology for a 
more^ detailed account of the processes of tissue preparation than 
can be given in this laboratory guide. 



10 



A LABORATORY GUIDE IN HISTOLOGY 



Cells. 



Plant Cells. With forceps remove a small piece of the epider- 
mis from a leaf of live-forever. Place it on a slide, add a drop of 

water, and cover 




CV%>ttV % 



Vv0s\ % w©vV*xy¥v\s» Va\}0W wis. \*.uW 2100. 



with a cover-glass. 
Study with low 
power. Observe 
the following: 

The large epider- 
mal cells with their 
irregular outlines. 
Search for the nuc- 
leus, spherical in 
form, and more re- 
fractive than the 
cytoplasm. The cytoplasm is granular and grayish in color. Look 
for vacuoles. Sketch a few cells carefully. See Fig. i. Using 
the high power, try to find cells in which there is a circulation of 
the protoplasm made evident by movements of the granules. Note 
the direction of the currents. Do you find the Brownian move- 
ment? Make a sketch and show by arrows the direction of the 
currents of cytoplasm. 

Study of Mitosis or Karyokinesis. The root tips of the onion 
or of Cypripedium were fixed in a chrom-acetic mixture, hardened 
in alcohol, and im- 
bedded in paraffin. 
Longitudinal sec- 
tions were made and 
fastened to the slides 
with Mayer's albu- 
min fixative. Take 
the slide given you, 
warm gently to soften 
the paraffin and place 
it in turpentine or 
xylol for several min- Fig 2 Mitosis fa the root . tip of PodophylluIT1 
utes until the paraffin peitatum. 




A LABORATORY GUIDE IN HISTOLOGY. 11 



^V^VACWs. 



is removed. Take through the grades of alcohol (absolute, 95%, 
80%, etc., two or three minutes in each) to water and stain deeply 
with Delafield's hematoxylin. Remove the excess of stain with 
acid alcohol, wash in tap-water, stain lightly with eosin, dehydrate, 
clear in oil of cloves, and mount in balsam. Study under high 
power and trace as many of the stages of mitosis as possible. 
Make sketches of cells showing the various forms of mitotic 
figures. See Fig. 2. 

Animal Cell Movements. Study under high power amoebae 
obtained by allowing a bit of fish, a fresh water mussel, or material 
collected from a pond, to 

decay in water. As soon /.£&\.>*^ /""\_^w>!&o^oSvm* 

as a slight film forms on 
the surface a drop should be 
placed on a slide covered 
and carefully examined. 
Search for small and nearly 
colorless bodies which are 
slowly chan ging their form . 
How does the animal 
move? Can you distinguish 
a clearer outer zone, the 

ectoplasm, from the more ^ ^ w& vA^V^~V^ 
granular endoplasm? Can 5 * ° J j 

you find a nucleus? A contractile vacuole? If so, study care- 
fully and determine its function if possible. Do you find a cell- 
wall ? Make a series of at least five sketches to show the changes 
inform. See Fig. 3. 

Animal Cells. Pieces of the ovary of a very young dog were fixed 
in bichloride of mercury or Flemming's fluid, hardened in alcohol, 
and imbedded in paraffin. Prepare your slide by carefully cleaning 

. with alcohol and spread a 

bVxOYAOk. very thin layer of Mayer's 

.tWoW. albumin fixative upon it. 

.x\\x\*AX«a. Avoid using too much fixative. 

W\XWw\xb. I* * s we ^ to add as little as 

^ ^. V \ f\ T*\ possible and spread it with 

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tov\xok£\\\l. M^UlA*,. 




12 



A LABORATORY GUIDE IX HISTOLOGY 



r-z — i tic 

section 



in this way as much as possible. Transfer the sectioi 
rm water bath to the slide by placing the slide under 
and lifting it from the water and carefully blot. (Cigarette paper 
makes splendid blotters for this purpose). Warm c = retully :ver z. 
gas flame or alcohol lamp until the paraffin begins to melt and re- 
move the paraffin with turpentine or xylol. Since dishes of xylol 
soon become charged with paraffin, as some one has suggested, it is 
a good plan to flood the slide with turpentine and drain on a blotter 
and then add xylol and follow with absolute alcohol, 95%, 80% , 
50%, and water, if stain used be an aqueous solution, otherwise use 
alcohol of the same grade as that used in the stain. Stain with 
Delafield's haematoxylin and remove the excess of stain with acid 
alcohol, or with a 2% aqueous solution of ammonia alum, and wash 
in water. Stain lightly in eosin, dehydrate, clear in oil of cloves 
for five or ten minutes, and mount in balsam. Study under low 
power, noting form of cell, relatively large nuclei, nucleoli, etc. 
Study nudes high power ani make sketches showing the general 
structure of a typical large animal cell. See Fig. 4. 



Epithelial Tissues. 



Squamous Epithelial Cells Unstained. S:r;__t the inside c: 
the lip or cheek with a clean scalpel and mount the scrapings in a 

X\ iv v \ * drop of physiolog- 

ical normal salt 
solution, or in the 
saliva, and study 
tinier -iz- power 
Note the shape 
and size of the 
cells and the shape 
and positions of 
the nuclei. Do any 
cells It = v e t ~ : 




KNAO c\ ~H*t\. 



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U*. ^r»\ 



toplasm clear or granular? 
leucocytes? Make sketche 

See FU-. f 

Stained Squamous Ce 
spreading material obtains 



"1.- 



d ^ t*^W\»\A^ nuclei? isthepro- 
>u find any salivary corpuscles or 
of several cells and label the parts. 

Make a cover-glass preparation by 
as above between two clean cover- 



A LABORATORY GUIDE IN HISTOLOGY 



13 



glasses and dry in the air. Fix by heat, passing quickly through 
the flame of a Bunsen burner or an alcohol lamp, and stain with 
hematoxylin and eosin. Dry carefully and mount in balsam. 
Study as above and sketch several cells. 

Squamous Epithelium of a Frog. A frog was kept in a glass 
jar for several days until portions of the epidermis were shed. The 
pieces were 

washed in wa- ^i^^iW^l V\A)A W*. 

ter and fixed &tttt&£^&m&> -.^V 




>x \ 



V\a,V>-V-6v\\\(\\uwiVY0CV * VSO. 



in alcohol, af- 
ter which 
they were 
stained in 



hsematoxylin 
and eosin, de- 
hydrated in 
alcohol, 
cleared with 

oil of cloves, or Eycleshymer's mixture. Take them from the oil 
and mount in balsam. Study the surface view of squamous epi- 
thelium thus prepared. Note the shape and size of the cells with 
their granular protoplasm and the intercellular cement. Do you 
find any intercellular spaces? Sketch a portion of the field show- 
ing form of the cells, nuclei, and their relations to each other. 
See Fig. 6. 

Stratified Squamous Epithelium. You will receive a section 
of the oesophagus of a cat which was imbedded in paraffin. Fasten 
to a clean slide with the fixative and after removing the paraffin by 

gentle warm- 
ing and treat- 
ing with tur- 
pentine, add 
xylol, abso- 
lute alcohol, 
alcohol, 
alcohol, 
alcohol 
water. 




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:\v<a<>\ 



AV 






l\\XA\. 



95% 

70% 
50% 

and 



11 



A LABORATORY GUIDE IX HISTOLOGY 



While it is desirable to use the grades of alcohol, it is not always 
done in general histological work. Stain in Delafield's hsematoxy- 
lin, excess of which may be removed with ammonia alum (2%), or 
with acid alcohol, wash in tap- water, and stain lightly with eosin. 
Dehydrate, clear in oil of cloves, and mount in balsam. 

Study the epithelial lining, first under low power and then 
under high power. Xote the layers of cells, columnar in the lower 
stratum and gradually becoming flattened as they approach the 
surface. Do the nuclei change in shape and structure? Do you 
note any change in the protoplasm of the cells of the different 
layers ? Make a sketch under high power to show the character 
and arrangement of the cells. See Fig. 7. 

Stratified Transitional Epithelium. Pieces of bladder were 
fixed in alcohol, imbedded in celloidin. and sectioned. Transfer 

the sections to 







_ XWMAevjOb 



V\(i\Ax(> k >a^\\xOV\^\\ w ^75>\^^^<, 



water and stain 
in Delafield's 
haemato xy lin 
and eosin. De- 
hydrate, clear in 
Eycleshymer's 
mixture and 
mount in balsam. 
Study the epi- 
* thelium under 

low and high power. How many strata of cells do you find ? Does 
the number vary in different parts of the section ? What is the form 
of the cell bodies of the deeper strata? Why do the cells 
van- in form ? Do the superficial cells have more than one nucleus ? 
Sketch a portion of the epithelium. See Fig. 8. 

Fresh Ciliated Epithelium. Carefully scrape the palate of a 
frog with a dull scalpel and mount the scrapings in normal salt 
solution. Study under high power noting the general form of the 
cell and the movement of the cilia. How do the cilia appear to 
move? Sketch a few T cells. 

Stained Ciliated Epithelium. Make a cover-glass smear of 
material secured as above, dry in the air and fix by passing rapidly 



A LABORATORY GUIDE IN HISTOLOGY. 



15 



through a flame two or three times, or expose to the vapor of 
formalin for ten minutes. Stain in hematoxylin and eosin, wash, 
dry, and mount in balsam. Study under high power and sketch a 
typical cell. 

Isolated Columnar Epithelial Cells. Macerate the mucous 
membrane of the small intestine of a cat in 33% alcohol for thirty-six 
hours. Tease a portion in dilute glycerin and cover. Tease 
another portion on a cover glass, dry and fix by passing over a 
flame, stain in hematoxylin and eosin, wash in water, dry thor- 
oughly, and mount in balsam. Study under high power. Is the 
protoplasm clear or granular ? Do the cells show striations ? Do 
they possess a cuticular border ? Note the shape and position of 
the nucleus. Sketch a few cells and label the parts. 



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0«<\. W\WW«, . 



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V\«AvUiLt oV(W& 



Columnar Epithelial Cells in Sections. The intestine of 
Necturus was hardened in alcohol, imbedded in celloidin, and 
sectioned. Stain in hematoxylin and eosin, dehydrate, clear in 
Kycleshymer's mixture, and mount in balsam. Study and sketch 
a few cells under high power. The cells of Necturus are very large 
and easily studied. See Fig. 8a. 



16 



A LABORATORY GUIDE IN HISTOLOGY 







Mesothelium and Endothelium. 

Mesothelium. Carefully remove the mesentery from the in- 
testine of a cat, rinse thoroughly in distilled water to remove any 
foreign substances that ma}' be present, and place in the dark in a 

i % solution of silver 
nitrate from ten to fif- 
teen minutes, or until 
it becomes somewhat 
opaque. Rinse in dis- 
tilled water and expose 
to the light in water or 
in 10% formalin until 
the silver is reduced 
giving a brown color. 
Rinse in water, stain in hematoxylin, dehydrate, clear, and mount 
in balsam. It is probably best to pin out the mesentery on thin 
sheets of cork until after the dehydration and clearing as it 
prevents the rolling and shriveling of the material. Study under 
high power, noting the shape and relation of the cells. Do }-ou 
find intercellular spaces ? Do you find stomata or pseudostomata ? 
Sketch a few cells carefully. See Fig. 9. 

Endothelium. The endothelial cells of the mesenteric capillar- 
ies are best shown by injecting the vessels of a small animal through 
the thoracic aorta. Chloroform a cat and when completely anaes- 
thetized, open the thorax and incite the heart so as to remove as 






much of the blood as possible. Inject from 75 to 100 c. c. of a 1% 
solution of silver nitrate, and after fifteen or twenty minutes inject 
from 100 to 150 c. c. of a io^c solution of formalin. After a few 
minutes open the abdominal cavity and expose the mesentery to 
the light. When the reduction of the silver has occurred, remove 




A LABORATORY GUIDE IN HISTOLOGY. 



17 



pieces of the mesentery, wash in water, study, and sketch under 
high power. See Fig. 10. 

Fibrous Connective Tissue. 



Unstained White Fibrous Tissue. Tease the portion of a ten- 
don of an ox given you in salt solution, cover and study under the 
low power and 
then under the 
high power. 
How are the 
fibrillse a r - 
ranged? Do the} 7 
branch or anas- 
tomose? Sketch. 
See Fig. n. Add 
a little dilute 
acetic acid ( i % ) 
at edge of cover- 
glass and draw 
it under with a 

piece of filter YvoAV\^\UVvV< 
paper. What is ^ 

the effect of the acid? Boil a piece of the tendon for some time. 
What is formed? What are the characteristics of white fibrous tissue? 




\Noxv\£*j. 






*<a \ v.s'bv^ o 



V \ wX> ov\©\0 * . 



Stained White Fibrous Tissue. A piece of tendon was hard- 
ened in alcohol, stained in borax-carmine, dehydrated, imbedded 
in paraffin, and sectioned longitudinally. Fasten to a slide, remove 
the paraffin with xylol, and mount in balsam. Are cells present? 
How are the fibers arranged ? Study and sketch under high power. 

Fresh Yellow Elastic Tissue. Tease a portion of the ligamen- 
tum nuchse of the ox in salt solution, cover and examine. Describe 
the general appearance of the fibers. Do they branch or anasto- 
mose? What position do the free ends assume? Are the fibers 
cemented into bundles? Are white fibers present? Add acetic acid 
as above. What is the result? Boil as above. What is formed as 
a result of the boiling? What are the characteristics of yellow elas- 
tic tissue? Sketch. See Fig. 12. 



18 



A LABORATORY GUIDE IN HISTOLOGY. 



Stained Yellow Elastic Tissue. A portion of the ligaraentum 
nuchas was hardened in alcohol, stained in borax -carmine, imbedded 
in paraffin, and sectioned longitudinally. Fasten to the slide, re- 
move the paraffin, and mount in balsam. Study and sketch under 
high power. 

Stained Yellow Elastic Tissue. The above was repeated for a 
cross section. Stud}- under high power, noting the grouping into 
bundles and the angular shape of the fibers. Do you find any cells? 
Sketch a small portion as seen under the high power. 







Areolar Connective Tissue. With your fine forceps remove a 
portion of the subcutaneous tissue of a cat or rabbit and place on a 
dry slide and, if the ends are drawn out and the center kept moist 
by breathing upon it, a thin film maj T be obtained which should be 
covered with a cover-glass having a drop of salt solution upon it. 
The tissue may be teased in the salt solution but it is less satisfac- 
tory than the above method. Stud}* under high power. How is 
the white fibrous tissue arranged? Note the arrangement of the 
elastic fibers. Sketch a portion of the field and label the parts. 
Add dilute acetic acid as above and note the effect upon the differ- 
ent tissues. Can you distinguish fixed connective tissue cells? 
Sketch a portion of the field so as to show the different tissues. 

Embryonic Connective Tissue. A portion of human umbilical 
cord was hardened in formalin, imbedded in celloidin, and 
sectioned. Stain your section in hseniatoxylin and eosin, dehy- 
drate, clear, and mount in balsam. Stud}' under high power. 
Note the branching connective tissue cells. Do they anastomose ? 



A LABORATORY GUIDE IN HISTOLOGY. 



19 



Do you find any intercellular substance ? Do you find fibers? 
Cross sections of blood vessels will be seen, but do not study them. 
Sketch a portion of the field showing form and relation of the cells. 
Fat Cells. The fatty mesentery of a dog was pinned to cork, 
fixed in alcohol, stained in hematoxylin, washed in tap- water, 
dehydrated, and cleared in oil of bergamot. Mount in balsam. 
Study under low power. The fat cells are clear and round or oval. 
How are they arranged ? What is their relation to the blood- 
vessels ? Sketch a group of the cells. Search for nuclei, using 
the high power. Sketch a typical cell. 

Cartilage. 

Hyaline. The larnyx of a rabbit was fixed in corrosive 
sublimate, hardened in alcohol, and imbedded in celloidin. Transfer 
the sections to water and stain in haematoxylin and eosin, 
dehydrate, clear, and mount in balsam. 

■v 




—to^vvAt. 



rO^n. - -- vvs. o\\ w . 






"V 




\ VC\.\2*. ^\»^viv*C,u<c\\uu«. ^xov^^A-O^^*- 



Study under high power. What is the character of the 
matrix ? What is the shape of the cells? Do they occur singly or 
in groups? Can you discern capsules around the cells? What is 
the structure of the perichondrium ? Note the change in the cells as 
you pass from the perichondrium toward the center of the cartilage. 
Make a sketch. See Fig. 13. 



20 



A LABORATORY GUIDE IN HISTOLOGY 



White Fibro=cartilage. A portion of interarticular fibro- 
cartilage was fixed in corrosive sublimate, hardened in alcohol, and 
imbedded in celloidin. Stain deeply in hematoxylin and then in 
Van Gieson's stain, dehydrate rapidly, clear in oil of origanum, and 
mount in balsam. 

Study carefully using the high power. What is the composition 
of the matrix ? Are the cartilage cells encapsulated ? Do you find 
any hyaline cartilage around the cells ? Why is this ? Sketch a 
portion of the section showing the structural relations. 

Elastic Fibro=cartilage. The epiglottis was fixed in corrosive 
sublimate, hardened in alcohol, and imbedded in celloidin. Stain 

with picro-carmineorwith the elastic 
tissue stain used by H. G. Harris. 
Stain for about ten minutes, wash 
one minute in i c /r nitric acid in 60% 
alcohol, dehydrate with 95% alco- 
hol, clear, and mount in balsam. 
How are the cells arranged ? Are 
they surrounded by hyaline carti- 
lage ? How may the elastic tissue 
What is the ar- 
of the elastic tissue? 
Sketch a portion as seen under 
high power so as to show the form 
and the relation of the tissues. 

Glycogen in Cartilage Cells. 

Treat /resk cartilage with dilute 
Lugol's solution, and glycogen may 
be found in the cartilage-cells, 
stained a peculiar brown color, 
usually described as mahogany 
brown. Study and sketch a few 
cells showing glycogen. 

Calcification and Ossification 
of Cartilage. The hand of a small human foetus was fixed in form- 
alin, dehydrated, and imbedded in celloidin. Stain with hema- 
toxylin and eosin, and mount in balsam. Study under low power. 
Note that there are well defined areas shown in a long bone. 




be recognized ? 



rangement 



Fig. 14. Long. Section of Meta- 
carpal Bones of a Fcetus; <a, hyaline 
cartilage; b, interosseous muscle; 
c t center of ossification. 



A LABORATORY GUIDE IN HISTOLOGY. 



21 



Where is hyaline cartilage found ? Do you find perichondrium or 
periosteum ? The areas should appear as follows if taken in order 
from the articular surface toward the center: (a) Hyaline or 
articular cartilage, (b) An area in which the cartilage cells are 
somewhat flattened and arranged in longitudinal rows, (c) An 
area in which the cartilage cells are greatly enlarged, perhaps dis- 
tending the capsules. (d) An area in which the cells seem 
shrunken and showing degeneration of the nuclei, (e) Area 
of ossification in which the primary marrow spaces are to be seen, 
as well as calcified cartilage trabeculse around which the osteoblasts 
are depositing layers of spongy bone. Sketch a portion so as to 
show a portion of each area in its proper relation to the other areas. 
See Fig. 14. • sj 

Bone. 

With the saw make thin transverse sections of a dry, clean 
metacarpal bone. Smooth one surface by rubbing on a fine file and 
then polish it on a fine hone. Using printer's paste or glue, fasten 
the sections on a smooth pine stick, allow the paste or glue to dry 
and then file the sections 
thin enough that the 
grain of the wood is 
easily seen through the 
section. Remove the 
sections by soaking in 
warm water, dry and pol 
ish on a fine hone, rub- 
bing until an examina- 
tion under low power 
shows the structures 
clearly. When perfectly 
dry mount on a slide in 
the following manner : 
Place a little very thick 
balsam on a slide and a 

little on a cover glass. 

tj ,,, y , , Fig. 15 Transverse Section Bone of Ox: a., 

xieat tne Slide and cover Haversian canal; b, bone of Haversian system; 

carefully and cool until c> intersystemic bone; d, lacunae. 

a film forms, place the section on the slide, cover quickly, and press 
cover down firmly. 



00 

- - 



A LABORATORY GUIDE IN HISTOLOGY. 



Study the transverse section first under low power and then 
under high power. The Haversian canals with the concentric 
lamellae of bone surrounding them are easily recognized. What is 
the nature of the matrix? Study the outer and inner circumferen- 
tial lamellae. Do you find Volkmann's canals? Note the inter- 
stitial or intersystemic bone between the concentric Haversian sys- 
tems. What is the position of the lacunae? Do the canaliculi 
anastomose? Do the canaliculi of the adjacent systems anastomose? 
Do the Haversian canals anastomose ? Sketch a portion under high 
power? See Fig. 15. 

Teeth. 



Sections of teeth may be made in the same manner as that 
described above for bone, if desired. The jaw of a young kitten 
or dog was placed in a mixture composed of 10% formalin 1 part, 

and 10% nitric acid 1 part, 
until the bone was decalci- 
fied when pieces were im- 
bedded and sectioned trans- 
versely and longitudinally. 
Fair sections may be made 
without imbedding as the 
pieces may be held in the 
microtome clamp. Stain 
the sections in haematoxylin 
and eosin, and mount in 
balsam. 

Study the transverse sec- 
tion of tooth and jaw under 
low power beginning at the 
center. What is the nature 
of the pulp? Surrounding 
the pulp and intimately 
connected with it by means 

of processes are the odon- 
Fig. 16. Transverse Section Jaw and Teeth toblast ceUs columnar in 
of a Cat: a, bone of a jaw; b, dental peri- 
osteum; c, cementum; d, dentine; e, pulp; torm. 
f, odontoblasts. Next in order is the den- 




A LABORATORY GUIDE IN HISTOLOGY. 



Xd 



tine composed of dentinal tubules lying in the intercellular substance 
or matrix. Do they branch ? 

Next in order is the granular layer of Tomes with its inter- 
globular spaces. 

Next in order is the cementum. How does it differ in appear- 
ance from the bone vou studied ? 



. ^llVOfAfetX. 



\Wuc0u1, 



► Ot\i 







\c\Cuv\a.. 



Next in order is the dental periosteum. What is its structure? 
Draw the section under low power. See Fig. 16. 

Study your longitudinal section of tooth and jaw in the same 
manner. Many sections will show the permanent tooth beneath the 
temporary. If so, study their relations. This section is designed 
to show the enamel and its relations and should be carefully studied. 
Make a sketch under low power showing the general relations. 
See Fig. 17. 

Muscle. 

Fresh Striated or Skeletal, (a) Tease a portion of the thigh 
muscle of afrog in physiological normal salt solution to isolate the 
fibers, and study under low and high powers. Do the fibers 



24 



A LABORATORY GUIDE IN HISTOLOGY. 



show striae? Do you find the sarcolernnia? Broken fibers usually 
show the sarcolemma. Add a little dilute (.7590 acetic acid 
at the edge of the coyer-glass and draw it under by holding 
filter paper at the opposite side. Where are the nuclei located? 
Do you find fibrillse ? Make a sketch to show the sarcolemma 
in a broken fiber. 

(b) Tease the muscle of Hydrophilus, which has been 
fixed in alcohol, in dilute glycerin and stud}- under high power. 
This preparation shows the details of striation better than most 
preparations. Sketch a few fibres. 

(c) Longitudinal Section Human Striated Muscle. Stain 
the section in haeniatoxylin and eosin and mount in balsam. 



SmfSk 



fti^u wWbwxvA,. 



m& 















£i »*v\ww\e,*\W«s. 






yai 



w^ c 



OA\ 



~ V ' 









•\ 



V\«l\VXV Auv*U*vV<W^A«, V. 100, 

Study under both powers and sketch a small portion under 
high power. Where are the nuclei located ? How are the 



fibers held together ? 



What makes the longitudinal striations ? 



Are the cells uni-nucleated or multi-nucleated ? 

(d) Longitudinal Section of Injected Muscle. The muscle 
of a cat injected with carmine-gelatin was imbedded in celloidin, 
after hardening in alcohol, and sectioned longitudinally. Study 
and sketch under low power showing the injected vessels and 
their relation to the fibers. 

(e) Transverse Section Human Muscle. Tissue was 
fixed and hardened in alcohol, imbedded in celloidin, and sec- 



A LABORATORY GUIDE IN HISTOLOGY. 25 

tioned. Stain the section in hematoxylin and eosin and mount 
in balsam. Study under low and high power. What is the 
position of the nuclei? What are Cohnheim's areas? Study 
the connective tissue of the muscle and the fibers. Sketch a 
small portion of the field under the high power. See Fig. 18. 

Cardiac Muscle, (a) Macerate cardiac muscle for twelve to 
twenty-four hours in fuming nitric acid of 20% strength. 
Wash out the acid in water and shake the muscle vigorously in 
a long test-tube, or tease gently on a slide, and study under 
high power. What is the shape of the cells? Do they branch? 
Are they striated ? What is the form and number of nuclei ? 
Sketch two or three cells. 

(b) The heart of a cat was fixed in bichloride of mercury, 
hardened in alcohol, imbedded in celloidin, and sectioned. Stain 
in hsematoxylin and eosin. Study under low and high powers. 
Do the bundles of fibers anastomose ? What is the position of 
the nuclei in cross sections ? 

Nonstriated Muscle, (a) Isolated Cells — Macerate and study 
as above for cardiac muscle. Make a sketch to show form and 
structure of the cells. 

(b) Study the arrangement and appearance of non- 
striated muscle in section by using the intestine of Necturus 
which has very large cells. Sketch a portion under high power 
showing longitudinal and transverse sections of muscle. See 
Fig. 8a. 

Blood. 

Fresh Human Blood. Obtain a small drop of blood by prick- 
ing the finger just below the root of the nail. The finger should 
be cleaned carefully and washed with alcohol and the needle 
should be sterilized in a flame. Place a drop on a clean slide 
and cover; mix a second drop with physiological normal salt 
solution and cover; place a large drop of blood on a third slide 
and allow clotting to commence before adding a cover-slip. 
Study the first preparation, noting that the red corpuscles tend 
to collect in rouleaux. Search for white corpuscles. Make 
drawings to show the rouleaux and the relative size of white 



26 



A LABORATORY GUIDE IX HISTOLOGY. 



and red corpuscles. Make a drawing of a red corpuscle on 
edge. 

Examine the second preparation in the same way, noting 
any differences in appearance. Do the red corpuscles collect in 
rouleaux? Wiry? Keep this second slide at 4o°C.,if possible, 
and tty to get a series of sketches showing amoeboid movement 
of the white corpuscles. 

The third preparation should be w T ashed gently to remove 
as many of the red corpuscles as possible. The section may 
now be stained with methylene blue and examined under a 
high power. How are the fibrin filaments arranged? Do they 



S ^OVv\Xvv\^^K\^OvU>;,N^. 



$» 




3C\W». 



k (XV"*\, 






j\s 



"*' 



-wXWs.0^A>w<O. 






^^\<\.^VVXXV\ fcXVU\> O S \0\OA VjyVXN &0^\tQ. 



^ 

stain? Sketch. The nuclei of white corpuscles will be stained 
a deep blue. 

Stained Human Blood, (i) Spread a drop of blood between 
two clean cover-glasses and quickly draw them apart and let 
the film dry. Place in forceps and fix by passing quickly through 
the flame. Stain in hematoxylin and eosin, dry carefully, and 
mount in balsam. The nuclei of the white cells stain with the 
hematoxylin while the eosin stains the protoplasm of the corpuscles 
and more deeply the eosinophile granules of the polynuclear cells. 
Examine with high power, noting the following forms of cells: 



A LABORATORY GUIDE IN HISTOLOGY. 27 

(a) Erythrocytes stained with eosin. 

(b) Small lymphocytes, mononuclear, nucleus relatively 
large and staining deeply, small amount of protoplasm sur- 
rounding the nucleus. 

(c) Larger mononuclear cells having a nucleus which 
stains less deeply than (b). 

(d) Transitional forms with U shaped nucleus. 

(e) Polynuclear cells with nuclei separate or lobulated 
and joined by fine threads of nuclear material. The nuclei 
stain quite deeply in most instances. Make drawing to show 
the various forms as seen under high power. See Fig. 19. 

(2) Make a preparation of blood on a clean slide by removing 
a drop with the end of a slide. Draw the second slide along on the 
first, holding it at an angle of 45 , thus leaving a thin film on the 
first slide. You will probably get a better preparation in this way 
than by using cover-glasses. Place the slides in formalin vapor for 
ten minutes and stain in methylene blue and eosin. Wash, dry, and 
mount in balsam. Study as above, noting the result obtained with 
this method of fixation as compared with the dry method. Prepar- 
ations may be made by treating the films with equal parts of ether 
and absolute alcohol for a number of hours, and then staining as 
above, or with haematoxylin and eosin, but, for general work, 
formalin vapor is greatly to be preferred. 

Frog or Turtle Blood. (1) Make cover-glass preparations, 
fix in the dry way, and stain with haematoxylin and eosin. Make 
drawings showing the nucleated red cells and the relative size of 
white and red cells. 

(2) Study the amoeboid movement of the white cells. Ring 
a cover-glass with vaseline and use it to cover a drop of blood mixed 
with a drop of normal salt solution. Find a white cell showing 
amoeboid movement and make six drawings, at two or three minute 
intervals, showing the changes in form. Can you distinguish ecto- 
plasm and endoplasm ? Are the pseudopodia clear or granular ? 

Bird Blood. Make and stain preparations of the blood of the 
English sparrow and study as above. It would be well to make 
drawings showing form and relative size of the erythrocytes of the 
various forms of blood that you have studied. 



28 



A LABORATORY GUIDE IX HISTOLOGY. 



Hsemin or Teichmann's Crystals of Human Blood. Place a 
drop of blood on a slide, add a small crystal or two of common salt, 

and after drying add a drop or two of glacial 
acetic acid, cover and heat gently over a 
flame until the acid boils. Cool and irrigate 
with water, or allow the acid to evaporate, 
and when perfectly dry mount in balsam. 
r? i \ • f v \ Study and make drawings of the brownish- 

Y\o^lo.\Wv^vU^aW black crysta i S) no ting carefully their form, 

See Fig. 20. 




size, 



and arrangement. 



Haemin Crystals of the Sparrow. Prepare as above and com- 
pare carefully with those from human blood. Show the difference 
and also resemblance by means of sketches. 



riasmin Crystals of Cat, Dog, and Rat. 

as above. 



Prepare and study 



Ha^min Crystals From Blood=stained Cloth. The portion of 
the cloth which you will receive has upon it dried blood which 
should be prepared as follows: Tease it on a slide in normal salt 
solution or in distilled water, add a few crystals of salt and set aside 
until the stain soaks out into the solution. Now remove the fibers 
and let the solution evaporate. The evaporation may be hastened 
by gentle warming; when dry add glacial acetic acid and finish the 
preparation as above. Examine carefully and try to identify the 
crystals. You will remember that this test is only to show that blood 
is present, as the crystals 
from pigeon's blood 
have much the same 
form and size as those of 
a mammal. Hsemin crys- 
tals are chlorides of 
hsematin, an iron com- 
pound found in the haem- 
oglobin. 

Study of a Blood= 
clot. Take a portion of 
the blood-clot given you, 
place it on a slide and \\o,.X\XA\^xovyv^\XV 



\Y\0A\-C*.' 



;v 







<V 



\W y / fij& tY ^^ * ^ u *^ • 




^C/A^OYWOA^ 



S 



\^YA^ 



>Wot,\A*,s. 






cover with a 30% solu- 



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o.vyo\aJ 



o\^Ti< 



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. 






A LABORATORY GUIDE IN T HISTOLOGY. 29 

tion of potassium hydrate until the clot softens when it is to be 
teased and examined for corpuscles. Can you identify them? 
Do they belong to bird, mammal, or amphibian? Hand a written 
report of your findings to the instructor. 

Red Marrow of Bones. From t-he red marrow of the bone of 
a cat or dog make several cover-glass preparations. Fix one by the 
dry method and stain in hematoxylin and eosin. Fix another in 
formalin vapor for ten minutes and stain in methylene blue and 
eosin. Fix one in equal parts of ether and absolute alcohol for 
twenty-four hours and stain in hematoxylin and eosin. Mount in 
balsam. Study under high power and make sketches of the various 
kinds of cells that you find. How do the marrow cells differ from 
leucocytes ? 

(a) Search for erythroblasts having large nuclei with dis- 
tinct chromatin threads, sometimes showing a tinge of color in 
the protoplasm. 

(b) Look for normoblasts having haemoglobin with glob- 
ular deeply stained nuclei and no chromatic filaments. What 
becomes of the nuclei as they are transformed into erythrocytes? 

(c) Look for mononuclear eosinophile cells and trans- 
itional eosinophile cells. 

( d) Search for polynuclear cells and lymphocytes. 

(e) Try to find giant cells with polymorphous and simple 
nuclei. See Fig. 21. 

Blood Vessels. 

The aorta of a dog was fixed in formalin, hardened in alcohol, 
and imbedded in paraffin. Fasten a section to a slide with albumin 
fixative, remove the paraffin, stain in hematoxylin and eosin, and 
mount in balsam. Study under low and high power. Three coats 
or tunics should be found from the outer to the inner as follows: 

(1) The tunica adventitia which is not very thick. What is 
its composition? How are the elastic fibers disposed? 

(2) The tunica media or middle coat consists largely of elastic 
tissue in concentric layers with very few smooth muscle cells. The 
tunica media is separated from the tunica intima and the tunica ad- 
ventitia by what are known as elastic limiting membranes. Do 
you find these membranes ? What is their structure ? 



80 



A LABORATORY GUIDE IN HISTOLOGY. 



(3) The tunica intima consists of a single layer of flat- 
tened endothelium, the nuclei of which project into the lumen 

v \ of the vessel, 




^^..ws&.ca, i<&w\&. 



and a sub-endothe- 
lial fibrous layer 
richer in cells in the 
inner than in the 
outer portion next 
to the internal elas- 
tic limiting mem- 
brane. What kind 
of fibrous tissue do 
you find in the in- 
tima ? Sketch a 
portion of the vessel 
and label the parts. 
See Fig. 22. 

Medium Sized 
Vein. Stain the sec- 
tion in haematox- 
ylin and Van Gie- 
son's stain and 
mount in balsam. Study carefully under high power and sketch 
a section. The tunica intima consists of three layers : 

( 1 ) A single layer of endothelial cells. 

(2) A layer of smooth muscle the bundles of which are 
somewhat separated by white fibrous connective tissue. 

(3) A fibro-elastic layer containing more white fibers 
than that of the artery. The tunica media, separated from the 
intima by an internal elastic membrane, contains circularly 
arranged muscle cells sometimes forming a continuous layer 
but often broken by strands of fibrous tissue. Compare with 
the media of an artery of the same size. The tunica adven- 
titia has an inner longitudinal muscular layer which is usually 
quite prominent. The adventitia is thicker than in the artery and 
has a greater amount of the white fibrous tissue. See Fig. 23. 

Capillaries. Anaesthetize a cat and open the thorax. Open 
the left ventricle and remove as much blood as possible. Now in- 
ject a 1 °/ solution of silver nitrate and after half an hour open the 






A LABORATORY GUIDE IN HISTOLOGY. 



31 



abdominal cavity, remove the mesentery and pin it out on sheets of 
cork and place in 10% formalin or 95% alcohol, and expose it to the 
sunlight until the tissue assumes a brownish tint. Dehydrate, clear 
in oil of ber^amot 



■V(\ 



ko\\\.iAv 



\.\XVA, 










t\t c^ 1 ^ 



& 



OowA.A^'i^' o 



X^. 



or in Kycleshymer ' s 
mixture, andmount 
in balsam. The 
nuclei may be 
stained with hem- 
atoxylin if de- 
sired. Study under 
high power search- 
ing for capillaries 
and the smaller ar- 
terioles, the endo- 
thelial cellsof which 

should be outlined \^^^^^^^^^^^^WA (kfc»%v&W 

in brown or black 

lines. What is the ^_— — «^ X\,\ 00 b-^A. 

shape of the cells? V' <*A?xW s U, v wU^. 
Is the outline regu- « * ■ . 

lar or irregular? Do you find any intercellular spaces? Make a 
drawing. See Fig, 10. 

Adenoid or Lymphatic Tissues. 

Diffuse adenoid tissue is found in the mucosa of the intestinal 
and respiratory tracts as ill-defined masses of lymphatic tissue. 
Search for it in sections of larynx, stomach, and colon. The retic- 
ulum is usually 
partially or en- 
tirely obscured 
by the very nu- 
merous lymph- 
oid cells. You 
may find it in 
places, however. 
Sketch a portion 
under the high 









)\\o^\A^^>. 



tY\VkVY\, 



32 



A LABORATORY GUIDE IN HISTOLOGY 



power. The section of the appendix of a rabbit is excellent for 
studying diffuse adenoid tissue, though nodules are fairly defined 
in the submucosa. 

Solitary Gland. Using the low power, search for solitary 
glands or lymphoid nodules in your section of the colon of a dog. 
Make a drawing to show the structure and relation to the surround- 
ing tissue. See Fig. 24. 

Thymus Gland. The thymus gland of a rabbit was fixed in a 
saturated solution of corrosive sublimate in normal salt solution, 
hardened in alcohol, and imbedded in celloidin. Stain in hsema- 







ttvAcx. 



toxylin and eosin and mount in balsam. Study first under low 
power noting the fibrous capsule from which septa pass dividing it 
into lobules. The lobules are likewise divided into secondary lob- 
ules within which are the follicles of adenoid tissue. Note the dif- 
ference between the cortical and medullary zones of the follicles. 
Search for the corpuscles of Hassal. What is their nature ? How 
may they be recognized under the microscope? Sketch a follicle as 
seen under high power. See Fig. 25. 

Lymph Gland. A lymph gland was fixed and hardened in 
alcohol and imbedded in paraffin. Fasten a section to the slide and 
stain in hsemotoxylin and eosin. Some authors recommend Flem- 
ming's solution and staining with safranin, especially for the study 
of the germ centers of the follicles. Study under low power, noting 
the connective tissue capsule. What elements enter into its com- 
position? Note that primary trabecule radiate toward the hilus 



A LABORATORY GUIDE IN HISTOLOGY. 



33 



from the capsule, thus dividing the gland into lobes. Note the sec- 
ondary trabeculse given off from the primary. Tertiary trabecule 
are given off from the secondary which anastomose in the lymphatic 
cords. Look for lymph sinuses between the capsule and cortical, 
follicles and along the primary trabecule. They are also found 
between the medullary cords and the trabecule. What is the nature 
of the medullary substance ? Draw under low power showing gen- 
eral relations. Study a follicle and its germ-center under high 
power and make a drawing showing the arrangement of the cells. 

Cells of Lymph Glands. Make preparations by drawing the 
freshly cut surface of a gland across the center of a clean slide. 
Fix in formalin vapor for ten minutes, or by heat, and stain in 
methylene blue and eosin. Dry and mount in balsam. Study under 
high power. Do you find lymphocytes? What varieties do you 
find ? Sketch a cell of each type found. 



The Spleen. 







(XvWvoW. 



Injected Spleen. The spleen of a cat was injected with car- 
mine-gelatin, hardened in alcohol, imbedded in celloidin, and sec- 



34 



A LABORATORY GUIDE IN HISTOLOGY. 



tioned. Dehydrate, clear, and mount in balsam. Study under 
low power. How do the arteries end ? Sketch under low power. 
See Fig. 26. 

Stained Spleen. Tissue from the spleen of a dog was fixed in 
a saturated solution of corrosive sublimate in normal salt solution, 
hardened in alcohol, imbedded in cellodin, and sectioned. Stain in 
hematoxylin and eosin and mount in balsam. Study under low 
power. What is the composition of the capsule? How is the 







frame-work of the gland formed ? What are the Malpighian cor- 
puscles? What is their relation to the arterioles of the splenic 
artery ? What is the nature of the splenic pulp between the Mal- 
pighian corpuscles ? Make a drawing showing the general struct- 
ural relations of the spleen. See Fig. 27. 

Study the spleen pulp under the high power. In the reticulum 
should be found the following varieties of cells: (1) erthrocytes; 
(2) a few nucleated erythrocytes; (3) giant cells; (4) leucocytes, 
especially the mononuclear variety; (5) cells containing pigment 
probably derived from the haemoglobin of broken-down red cells. 



A LABORATORY GUIDE IN HISTOLOGY. 



35 



Nucleated Red Corpuscles in Spleen. The method of Dr. E. 
T. Williams gives good results. Take the fresh spleen of a hog 
and from the outer edge cut wedge shaped pieces which are drawn 
very lightly across the center of a clean slide making a thin smear. 
Fix for one minute in the following fluid : 

Corrosive sublimate .78 gram. 
Sodium chloride .28 gram. 
Distilled water 30 c. c. 

Stain in an aqueous solution of hematoxylin, and counter- 
stain in eosin. Dry and study without a cover glass under high 
power. These preparations may be permanently mounted in bal- 
sam. Sketch a few erythrocytes, and erythroblasts if they are 
present. 



The Tongue. 



The apex of the tongue of a rabbit was fixed in bichloride of 
mercury, hardened in alcohol, and imbedded in paraffin. Fasten the 
section to the slide, remove the paraffin, stain in hsematoxylin and 
Van Gieson's stain, dehydrate rapidly, clear in oil of cloves, and 
mount in balsam. 

Study under low power. What forms of papillae do you find? 
What kind of epithelium covers them? Do the cells covering the 
papillae meet at the 



edges or overlap ? Do 
you find a connective 
tissue within the pa- 
pillae? Do you find 
any glands ? Where 
are they located? Are 
they serous or mu- 
cous ? Study the mus- 
culature of thetongue. 
Is the muscle smooth 
or striated ? How are 
the muscles arranged? 
Do you find any 
nerves? Sketch as 
seen under low power. 




„- \>\o0O-V>tfcSSA. 

_LOWY\\\S«>\A«, COX*. 



.-\aastvY\evv\ 



V, 



°s 



X%. \\kv\o^\©\*<\ \ wa\\\<X. 



36 



A LABORATORY GUIDE IN HISTOLOGY. 




>\W\A\ 






V\ o^ 1 \\ % . Vo^W uQ o dC\ o *V <*£** - 



Fungiform Papillae. Portions of the tongue of the dog con- 
taining fungiform papillae were fixed and hardened in alcohol, im- 
bedded in celloidin, and 
sectioned. Stain in hema- 
toxylin and eosin, dehy- 
drate, clear and mount. 

Study under low power. 
Note the connective tissue 
core and epithelial covering 
of the papillae. Do you 
find any taste-buds in the 
epithelium of the papillae or in that surrounding it ? Sketch papil- 
lae as seen under low power. See Figs. 28 and 29. 

Taste Buds. Portions from the tongue of the rabbit contain, 
ing foliate papillae were fixed, hardened, imbedded in paraffin, and 
sectioned at right angles 
to the folds. Stain in 
haematoxylin and eosin, 
after fixing to the slide 
and removing the par- 
affin, clear in oil of cloves 
and mount. Study first 
under low power. What 
is the location of the 
taste-buds ? Do they 
extend through the epi- 
thelium ? What type of 
glands do you find? 
What are they called? 
Sketch a portion as seen under low power. See Fig. 30. 

Study the taste buds under high power. What is their form ? 
Are they completely surrounded by epithelium ? Sketch and show 
as much of the structure as you can. 

Circumvallate Papillae. Portions of the tongue containing 
circumvallate papillae were hardened in alcohol, imbedded in cel- 
loidin and sectioned. Stain in haematoxylin and eosin, clear in 
Bycleshymer's mixture and mount in balsam. Study under low 
and high power as above and sketch as seen under low power, show- 
ing the structural features. 




Fig. 30. Transverse section of the Foliate Pa- 
pillae of a Ribbit : a., taste-buds; b f papilla; c, 
glands; d, muscle. 



A LABORATORY GUIDE IN HISTOLOGY 



37 



Alimentary Tract. 

(Esophagus. The oesophagus of a dog was fixed in bichloride 
of mercury, hardened in alcohol, and imbedded in celloidin. Stain 
the section in hematoxylin and eosin, dehydrate, clear in Eycle- 
shymer's mixture, and mount in balsam. 




«.^ 



'\VW/lSk 



\\>w\. 



\AtV 



Xe\w<»v^y 






w\utov«v. 



^EEFC^r^S^^^rr^r V-tWCWOOC VAvV^oW 



.\o WOk, YAWS VW . 



'CO Y\V\ Ct\"\\> fcYvSS^t., , 



Yvc^VC^w^cvOaMi o\r (\T3i o a >L 5> 0. 



Study under low power. What kind of epithelium do you 
find ? Do you find a muscularis mucosae ? Study the structures 
found in the tela submucosa. What kind of glands do you find ? 
Study the muscular layers. Is the muscle striated or non-striated ? 
Sketch under low power and show the parts and general relations. 
See Fig. 31. 

Cardiac Stomach at its Junction with the (Esophagus. 

Stain in hematoxylin and Congo red and mount in balsam. Study 



38 



A LABORATORY GUIDE IN HISTOLOGY 



Note changes in epithelium from 

Do you find glands in the tela sub- 

What kinds of muscle do 3'ou find 

Make a drawing of the epithelium at 



under low and high power. 

stratified to simple columnar. 

mucosa ? If so. what kind? 

and how are they arranged ? 

the junction of the oesophagus and stomach. 

The Stomach with Blood Vessels Injected. After injection 
through the aorta with carmine-gelatin, the stomach of a cat was 

hardened in alcohol, imbedded in cel- 
loidin, and sectioned. The sections 
are in clearing oil from which you 
will mount them in balsam. Study 
and sketch under low power showing 
the arrangement of the blood vessels. 
See Fig. 32. 

Cardiac Glands. Portions of the 
cardiac stomach of a dog were fixed 
in corrosive sublimate solution, hard- 
ened in alcohol, and imbedded in paraf- 
fin. Fix sections to slide, remove 
paraffin, and stain in hcematox5 r lin and Congo red. Clear in oil of 
cloves and mount in balsam. 




Fig. 32 Section of the Mucosa 
of a Cat's Stomach injected. 



^ — 0w\ o^\s. 



&\\^c\tA\ 



oouC 




UikouV 



\XW\W\j. 



^vO^Vb.V^oc^\^V^vx>^&^ocife^\ovAU^. 



Studj T under low power to get the general structural relations. 
What type of gland do you find ? What is the nature of the tela 



A LABORATORY GUIDE IN HISTOLOGY. 



39 



submucosa ? Do you find any glands in it ? How are the muscular 
tissues arranged ? Sketch a portion as seen under low power. 

Study the glands under the high power noting the chief or 
peptic cells and the parietal cells, oval in shape, with prominent 
nuclei, and stained with the Congo red. Sketch. See Fig. 33. 

Pyloric Glands. Portions of stomach from the pyloric region 
were fixed in corrosive sublimate, hardened in alcohol, imbedded in 



XVWW)(UA C^<&V\ 




celloidin, and sectioned. Stain in hematoxylin and eosin, dehy- 
drate, clear in oil of bergamot, and mount in balsam. 

Study under low power. How do the ducts of the pyloric 




g. tow*\*,c 



tS\\>S, \ 



V»*\M. 



glands differ from those of the cardiac glands ? How do the secre- 
tory tubules differ ? Compare the muscular layer with that of the 



40 



A LABORATORY GUIDE IN HISTOLOGY 



cardiac end. Do you find any goblet cells in either cardiac or 
pyloric section-? Sketch a portion under the high power to show 
the glandular epithelium and compare with the epithelium of the 
cardiac stomach. 

Duodenum. Portions of the duodenum of a dog were fixed in 
bichloride of mercury, hardened in alcohol, imbedded in celloidin, 

and sectioned. Stain 
in hematoxylin and 
eosin and mount. 
Stud}- under low 
power noting the coats 
and general relations. 
Do you find villi? 
Xote the crypts of 
Lieberkuhn and the 
glands of the tunica 
mucosa. Find Brun- 
ner's glands in the 
tela submucosa. Are 
the>* serous or mu- 
cous? Are they sim- 
ple or compound ? 
Tubular or saccular? 
Make a drawing using 
the low power so as 
to show the general 
structure. See Fig 34. 




Fig. 34. Human Duodenum: a. solitary lymph nod- 
ule ; b. diffuse adenoid tissue ; c. Brunners glands : 
d, glands of Lieberkiihn. 



Injected Ileum of a Cat. The injection mass used was car- 
mine-gelatin and it was injected through the abdominal aorta. The 
intestines were hardened in alcohol, imbedded in celloidin, and 
sectioned. The sections are in clearing fluid and are to be mounted 
in balsam. Study and sketch under low power. See Fig. 35. 

Small Intestine Stained. Portions of the ileum were fixed in 
bichloride of mercury, hardened in alcohol, imbedded in celloidin. 
and sectioned. Stain in hematoxylin and Yan Gieson's stain, de- 
hydrate quickly, clear in Eyeleshymer's mixture, and mount in bal- 
sam. 

Study under low power. What is the shape and structure of 



A LABORATORY GUIDE IN HISTOLOGY. 



41 



the villi? Note the glands of 
Lieberkiihn. Do they extend 
through the mucosa? What kind 
of epithelium covers the villi ? 
Are goblet cells present ? Study 
the muscularis mucosae. Note 
the vessels of the tela submu- 
cosa. Look for nerves in the 
submucosa and between the 
muscular layers. Do you find 
adenoid tissue ? In what form 
does it occur ? Note the tunica 
muscularis and the tunica serosa. 
What is the structure of the 
tunica serosa ? Sketch a portion 
under the low power. 

Colon. Portions of the colon 
of a dog were fixed in bichloride 





Fig. 35a. Colon of Man : a, glands of Lieberkiihn; 
b, tela submucosa ; c, circular muscle layer ; d, longitud- 
inal muscle layer; e, muscularis mucosae; f, blood-vessel. 



Fig. 35. Injected Intestine of a Cat. 



of mercury, hard- 
ened in alcohol, 
and imbedded in 
paraffin. Fix the 
section to the 
slide, remove the 
paraffin, and stain 
with haematoxy- 
lin and eosin. 
Mount in balsam 
and study under 
low power. Are 
villi present? 
What glands are 
found in the tun- 
ica mucosa? What 
kind of epithelium 
do you find? Are 
goblet cells pres- 
ent? Do you find. 



42 



A LABORATORY GUIDE IN HISTOLOGY. 



diffuse adenoid tissue ? Are solitary glands present ? How is the 
muscular tissue arranged in the tunica muscularis ?- Sketch to 
show general structural relations and draw a gland of Lieberkiihn 
under the high power. See Fig. 35a. 

Vermiform Appendix. Portions of the appendix of a rabbit 
were hardened in alcohol, stained in Delafield's haematoxylin, and 
imbedded in paraffin. Fix to the slide, remove the paraffin with 
turpentine and xylol, and mount in balsam. Study and sketch 
under low power noting carefully the structural relations. 



Digestive Glands. 



Parotid. — Serous Type. Small pieces of the parotid gland of 
a dog were fixed and hardened in absolute alcohol, and imbedded 
in paraffin. Fix the section to the slide, stain in hematoxylin and 



\Y\\*/\o\i\A& 




V <J.Ov\v\.d 



<AsV« 



\ ■>**«.- 



,-Ckt,\YVVA , a 1 



fei » )ji' ! 

eosin, and mount in balsam. Study under low power and note the 
lobules which are held together by connective tissue. Of what 
structures are the lobules composed ? Do you find any intralobular 
ducts? Study the acini under the high power. Is the protoplasm 
clear or granular ? Are the cells in the resting or active condition ? 
Do you find a basement membrane ? Sketch a portion under the 
low power to show the general relations and a few of the acini 



A LABORATORY GUIDE IN HISTOLOGY. 



48 



under the high power to show the epithelium of the acini and a 
duct. See Fig. 36. 

Submaxillary of the Dog. — Mucous Type. The tissue was 
fixed in Flemming's solution, and imbedded in paraffin. Fix the 
section to the slide, and stain in Delafield's hematoxylin and 
Congo red. Dehy- 
drate, clear, and 



mount in balsam. 
Study first under 
low power noting 
that the general 
















OvAiofUk-vyW 



structure is very 

similar to that of 

the parotid gland. 

Study the acini un- n 

der hieh power Is ?J 3 1" ^ UMW *** ^«.*o** v\tvu ^A\*.vAa>\ OvaW*m\Wx^. 

a basement membrane present ? Are the cells clear or granular ? 
What is the position of the nucleus ? Look for the crescents of 
Gianuzzi or the demilunes of Heidenhain. What relation do they 
bear to the mucous cells of the acini ? Are the cells of the cres- 
cents clearly defined? Sketch a small portion showing a duct, 
mucous acini and the crescents. See Fig. 37. 

The Pancreas. Pieces of the pancreas of a dog were fixed in 
bichloride of mercury, hardened in alcohol, and imbedded in paraf : 

\ fin. Fix the sec- 

tion to the slide, 
remove the par- 
affin, stain, and 
mount in bal- 
sam. 

Study under 
low power not- 
ing that the 
structure and 
general appear- 
ance is very sim- 
ilar to that of the 
parotid gland. 







Vj av\fi«.v\\UWb. 



v xowa,, 






44 



A LABORATORY GUIDE IN HISTOLOGY 



Study the aciui under the high power. Is the protoplasm clear 
of granular ? What is the position of the nucleus ? Is a basement 
membrane present ? Do you find the centro-acinal cells ? Do you 
find the areas of Langerhans between the acini of the gland ? How 
do they appear in contrast with the acini ? Do you find any traces 
of ducts in them ? Sketch an area of Langerhans with the sur- 
rounding acini as seen under high power. See Fig. 38. 

The Liver with Blood= Vessels Injected. The liver of a dog 
was injected through the portal vein with carmine-gelatin, hardened 

in alcohol, imbed- 
ded in celloidin, 
and sectioned. The 
sections are in 
clearing oil and 
should be mounted 
in balsam. 

Study under low 
power noting the 
lobules and their 
arrangement. Be- 
tween the lobules 
are the interlobular 
veins. Of what are 
they branches? 
Study the capilla- 
ries of the lobule 
noting that they 
originate from the 
interlobular and 




Fig. 39. Dog Liver Injected: a., interlobular veins; b f 
vena centralis lobulae ; c, branch of portal vein. 



that they pass into the capillaries of the vena centralis lobulae, the 
beginning of the hepatic system of veins. It is difficult to de- 
termine where the capillaries from the portal vein cease and those 
of the hepatic vein begin. By searching the section you should be 
able to find where a vena centralis lobulse passes into a sublobular 
vein. Sketch under low power. See Fig. 39. 

Injected and Stained Liver. Portions of liver as injected 
above were stained in Delafield's hematoxylin, imbedded in celloi- 
din, and sectioned. Remove the sections from the clearing oil and 



A LABORATORY GUIDE IN HISTOLOGY. 



45 



mount in balsam. Study as above, noting the relation of the 
hepatic cells to the capillaries, and sketch a small portion under 
high power. 

Stained Pig Liver. Small pieces of pig liver were hardened 
in absolute alcohol and imbedded in paraffin. Fix the section to 
the slide, remove 
paraffin, and 
stain in haema- 
t o x y 1 i n and 
eosin, clear in oil 
of cloves, and 
mount in bal- 
sam. 

Study the lob- 
ules and general 
structure under 
low power. The 
pig' sliver is well 
adapted for the v ^v\\v\oV\i 

study of the gen- ?^ 4o.TU<y* &«, $WU,\-.W\i oV^U 

eral structure 

since the lobules are very distinct. Find a portal canal containing 
a bile duct, hepatic artery, and portal vein. What is the position 
of the canal ? Study the capsule of Glisson. Do you find lym- 
phatic vessels in the canals? Draw a portal canal with portions of 
the adjacent lobules as seen under low power. See Fig. 40. 




LV>\\* &v.\«A. 

caa*v\e o\ vs\\v»ov\- 



,(XV \}4aW. 



-AVtVXftO. 



r 




, C<X^\\\c*\'\ts. 



Impregnated Liver. Oppel's 
Method. Pieces of liver were 
placed for three days in a solu- 
tion consisting of four parts 3 °/ c 
solution of potassium bichro- 
mate, and one part of a 1 % so- 
lution of osmic acid and then 
placed in a ^ to 1 % solution of 
silver nitrate where they re- 
mained several days, after which 
they were rapidly imbedded in 
T\a.HI. &At Cc^vWfcwts <v^^WWy<\o. celloidin, sectioned, and you will 



(A ^t\. 



46 



A LABORATORY GUIDE IN HISTOLOGY. 



mount and study them in the clearing oil. The} 7 may be mounted 
in hard balsam. The bile capillaries are stained black. Study 
their relations to the cells and sketch under high power. See 
Fig. 41. 

The Kidney. 

Kidney Macerated to Show Tubules. Place strips of the 
kidney of a cat or other animal in 25 to 30% hydrochloric acid for 
twenty four hours after which wash one hour in running water. 



|^..u\c\^\o}v\&YV \}06u. 




v n\ooq oV iAiv\«/. 



Tease carefully and examine in 50% glycerin. The tubules will 
be broken up somewhat but by studying the preparation carefully 
under low power the parts of the tubule may be found. Sketch 
and label as many parts as you can recognize. See Fig. 42. 

Kidney of a Cat Injected. The kidney of a cat was injected 
through the renal artery with carmine-gelatin, hardened in alcohol, 
imbedded in celloidin, and sectioned. Remove the sections from 
the clearing oil and mount in balsam. 

Study under low power. Do you find portions of the arterial 
arch ? Note the straight arterise rectae extending into the medulla. 
Trace an interlobular artery from the arch into the cortex. Study 
the vasa afferentia as they terminate in the capillary loops or glom- 
eruli. Trace the vasa efferentia as they leave the glomeruli and 



A LABORATORY GUIDE IN HISTOLOGY. 



47 



form capillary 
net works about 
the convoluted 
portions of the 
tubuli uriniferi. 
Study the for- 
mation of the 
capillary net- 
work in the su- 
perficial part of 
the cortex . 
Draw under low 
power showing 
as many of these 
points as you 
can. See Fig. 43. 

Stained Kid= 
ney. A portion 
of the kidney of 
a cat was fixed 
in Carnoy ' s 
fluid for about 
five hours, the 
fluid was then 




Fig. 43. Injected Kidney of a Cat x70. 



washed out with absolute alcohol and the tissue was imbedded in 
celloidin, sectioned transversely, stained in iron-alum-haematoxylin, 
counterstained in acid-fuchsin, and the sections are now in clearing 
oil from which they are to be mounted in balsam. 

Study under low power noting the capsule and the division 
into cortex and medulla. 

Locate the medullary rays extending into the cortex from the 
base of the pyramid. Of what are they composed ? Locate the 
columns of Bertini. What are they and why are they present ? 
What is the labyrinth ? Study the pyramids. Why do they have 
a striated appearance ? Look for blood capillaries. Study the 
glomeruli. What is the relation of the glomerulus to Bowman's 
capsule ? Sketch under low power to show the general structural 
relations. See Fig. 44. 

Study the glomeruli under the high power. Study the differ- 



48 



A LABORATORY GUIDE IN HISTOLOGY. 




ent portions of the tu- 
bules under high power 
noting the variations 
in the character of the 
epithelium and sketch 
cells from each part 
to show the nature of 
the epithelium. Do you 
find a basement mem- 
brane? Study the cap- 
sule under high power. 
What is its composition? 
Notice the intertubular 
connective tissue, rela- 
tively small in amount 
in the normal kidney. 
Sketch a glomerulus and 

Fig. 44. Human Kidney: a., glomerulus; b t a portion of the SUr- 
medullary ray ; c, convoluted tubules ; ^ Bow- roundin " tissue as Seen 
man's capsule. . 

under high power. 

Longitudinal Section of the Kidney of a Foetal Pig. The 

kidney of a foetal pig was hardened in alcohol, imbedded in celloi- 
din, and sectioned. Stain in haematoxylin and eosin, clear in 
Eycleshymer's mixture, and mount. Study as above, noting the 
development of the tubules and the relation of the glomerulus to 
Bowman's capsule. Sketch a portion. 

Suprarenal Body. 

The suprarenal body of a rabbit was hardened in alcohol and 
imbedded in paraffin. Fix the section to the slide, remove the 
paraffin, stain in hsematoxylin and eosin, clear in oil of cloves, and 
mount in balsam. 

Study under low power. Study the capsule and the septa 
passing into the body. Note the division into cortex and medulla. 
Study the cortex noting the zones of which it is composed. The 
outer zone is known as the glomerular zone since the cells are in 
groups separated by septa derived from the capsule. The middle 
zone has the cells arranged in columns or bundles of cells separated 
by the connective tissue and is called the fascicular zone. The 



A LABORATORY GUIDE IN HISTOLOGY. 



49 



inner zone is composed of anastomosing cords of cells and is known 
as the reticular zone. Note that the medulla is composed of strands 
of cells usually pigmented and irregular in shape. What is the 




^ 



*\M. 



|Lw<^ 



XOWCK 



\ v 



>t^C\. 






«.0V^% o^ u\\». 

nature of the spaces between the strands of cells ? Study the 
blood vessels and nerves of the gland. Sketch a section under low 
power showing general structure. See Fig. 45. 

Bladder and Ureter. 

Collapsed Bladder. Pieces of the bladder of a cat were fixed 
and hardened in alcohol, imbedded in paraffin, and sectioned. Fix 
the section to the slide, remove the paraffin, stain in hematoxylin 
and eosin, clear in oil of cloves, and mount in balsam. 

Study under low power. What kind of epithelium do you 
find ? How many layers of cells are present ? How do the cells 
of the various layers differ in form, and why do they so differ ? Is 
diffuse adenoid tissue to be found in the tunica mucosa ? Do you 
find glands in either the tunica mucosa or the tela submucosa? 



50 



A LABORATORY GUIDE IN HISTOLOGY. 




How many muscular layers do you find in the tunica muscularis? 

Is the muscular tissue regularly or irregularly disposed ? Do you 

find a tunica serosa ? Study the sec- 
tion under high power and identify 
the structures fully, asking help of 
the instructor if necessa^, and sketch 
a portion under the low power in order 
to show the relative thickness and 
relations of the layers. See Fig. 46. 

Distended Bladder. The bladder 
of a rabbit was distended with the fix- 
ing fluid and then placed in a quantity 
of the same fluid until fixed and hard- 
ened after which it was imbedded in 
paraffin, sectioned, and fixed to cover 
glasses. Remove the paraffin with 
turpentine and xylol, stain in hema- 
toxylin and Van Gieson's stain, de- 
hydrate quickly, clear, and mount. 
Study as above and compare the sec- 
tions carefully noting the principal 
points of difference in the appearance. 

Ureter Near Pelvis. Tissue was 
prepared as for the section of collapsed 
bladder and you will prepare and study in the same manner. 

Ureter Near Bladder. The tissue was treated as above and 
the sections are to be stained and mounted. Compare the sections 
of ureter, especially the musculature. What kind of connective 
tissue do you find ? 

Male Reproductive Organs. 

Testis. The testis of a rat was fixed in Flemming's solution, 
imbedded in paraffin, sectioned transversely, and fixed to cover- 
glasses. Remove the paraffin, stain in hematoxylin and eosin, de- 
hydrate, clear in oil of cloves, and mount in balsam. Study under 
low power, noting first the capsula fibrosa or tunica albuginea which 
surrounds it. Trace the trabeculse from the capsule into the gland 



Fig. 46. Collapsed Bladder of a 
Rabbit: a, connective tissue of 
tunica propria ; b, transitional 
epithelium; c, muscle in longitu- 
dinal section; d, muscle in trans- 
verse section. 



A LABORATORY GUIDE IN HISTOLOGY. 



51 



substance. Some 
fatty tissue may be 
found outside the cap- 
sule. Study the sec- 
tions of the tubuli 
seminiferi, some of 
which are cut trans- 
versely and some 
obliquely, noting the 
layers of epithelial 
cells forming the 
walls. Note the jepi- 
didymis at one side of 
the section. How is 
the canal of it lined ? 
Fat globules may be 
found stained black 
with the osmic acid. 
Sketch a portion un- 



*-^~< 




m 







j a 






Fig. 48. Single Tubule Rat's Testis highly 
magnified. The lumen of the tubule is seen at 
the center and the sperm cells may be seen in 
radial rows. 



Fig. 47. Testis of a Rat under low power : a., 
tubule cut longitudinally ; h, tubule cut transverse- 
ly; c, intertubular connective tissue. 



der low power to show 
the general relations. 
See Fig 47. 

Study the tubules 
under high power. By 
moving the section tub- 
ules in different stages 
of development may be 
found. Study the inter- 
tubular tissue carefully. 
Do you find a distinct 
basement membrane? 
What is the nature of 
it? Within the mem- 
brane you find the layer 
of spermatogones and 
the bases of the support- 
ing cells or cells of Ser- 
toli. The second layer 



52 



A LABORATORY GUIDE IN HISTOLOGY 



is that of the sperm mother-cells or spermatocytes. Next in 
order are several laj^ers of smaller cells, the spermatoblasts 
or spermatids, which become sperm cells without further division. 
Can you find spermatids that have fused with the cells of Sertoli ? 
Study various tubules very carefully and sketch several to show as 
man}^ of the stages in development as possible. See Fig. 48. 

Testis. Compare with the above sections prepared as above 
and stained in iron-alum-hsematoxylin. Study the tubules of this 



' 7 - \ ' A ■ ■" 


' * k JE!F/* •>■ ■:"'•< > 






1 %0*i 




jDH^HBlQSiflHI^^HP'Z/'VJl 





Fig. 49. Transverse Section of the Penis of a Dog : a, urethral lumen; 
b, epithelium; c, corpus spongiosum surrounding the urethra; d, corpora 
cavernosa showing some ossification; e, tunica albuginea; f, blood-vessel. 

section under high power as this stain is very satisfactory for the 
nuclear structures. 

Penis. The penis of a dog was fixed and hardened in alcohol, 
imbedded in celloidin, and sectioned transversely. Stain in hsema- 
toxylin and Van Gieson's stain, dehydrate rapidly, clear in Kycle- 
shymer's mixture, and mount in balsam. 



A LABORATORY GUIDE IN HISTOLOGY. 53 

Study first under low power noting the three masses of erectile 
tissue. The corpora cavernosa have a strong, dense tunica albu- 
ginea surrounding them. The corpus spongiosum surrounds the 
urethra. Note the septa and trabecule derived from the tunica 
albuginea. What tissues are found in the sheath and septa ? Do 
the spaces formed by the septa and trabeculae communicate with 
each other ? Are the spaces lined with endothelium ? Note the 
blood vessels. Study the urethra. What kind of epithelium in it? 
Do you find any glands ? Study the blood vessels of the submu- 
cosa and the muscular tissue of the urethra. Sketch as seen under 
the low power and label the parts. See Fig. 49. 

Prostate Gland. The prostate gland of a dog was fixed and 
hardened in alcohol, imbedded in celloidin, and sectioned. Stain in 
hematoxylin and eosin, dehydrate, clear in Eycleshymer's mixture, 
and mount in balsam. 

Study under low power. Notice the structure of the capsule 
noting the large proportion of smooth muscle in it. Study the tra- 
becular framework. What type of gland do you find? What kind 
of epithelium lines the tubule ? Is it a serous or mucous gland? 
Is a basement membrane present? Find the duct emptying into 
the urethra. What kind of epithelium forms the lining of the 
duct ? Sketch a portion under low power to show the general 
structural relations and a portion of a gland as seen under high 
power. 

Cowper's Gland. Portions of the gland were fixed, hardened, 
imbedded in celloidin, and sectioned. Stain in hematoxylin and 
eosin, and mount in balsam. Study the general structural features 
under low power. What type of gland as to form ? As to secre- 
tion ? Are crescents of Gianuzzi present ? What kind of epithe- 
lial lining do the ducts possess ? Look for larger ducts lined by 
two or three layers of cells. Sketch a portion of the gland as 
seen under high power. 

Female Reproductive Organs. 

Ovary. The ovary of a dog was fixed in Flemming's solution, 
hardened in alcohol, and imbedded in paraffin. Fasten the section 
to the slide, remove the paraffin, stain in hematoxylin and eosin, 
clear in oil of cloves, and mount in balsam. 



54 



A LABORATORY GUIDE IN HISTOLOGY. 




tft^\< 



(XX v V 



_.tv\©ob\A. 



.\v 



_ x\wv^h> 






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b\^ 



Study first using the low power. Note the division into the 
cortical and medullary portions. The larger vessels are found in 
the medulla. Study the germinal epithelial layer. The framework 

of the ovary is known as the 
stroma. What is its composition? 
In the cortex you should find 
the Graafian follicles in the dif- 
ferent stages of development. 
The simple primitive follicle has 
but a single layer of epithelial 
cells surrounding the egg - cell 
and surrounded by stroma. Find 
such follicles. The fully de- 
veloped follicle has a rather 
^vkvAX^OQ. dense stroma, the theca folliculi, 
surrounding it and instead of a 
single layer of cells lining it, it will have several layers forming the 
membrana granulosa. Can you distinguish the fibrous tunica ex- 
terna of the theca ? Note that this layer blends closely with 
the tunica interna 
which is more vas- 
cular than the ex- 
terna and has more 
cells. One portion 
of this membrana 
granulosa becomes 
thickened forming 
the discus proliger- 
us surrounding the 
egg-cell. Some of 
the cells have dis- 
appeared forming 
the antrum or cav- 
ity of the follicle, 
containing the liq- 
uor folliculi. What 

causes the precipi- Fig. 51. Ovary of a Dog: a, stroma; 6, blood-ves- 
tate found in the sels; c. empty Graafian follicles; d, membrana granu- 
losa ; e, antrum of the follicle ; f, discus proligerus ; 
antrum in the sec- g , ovum. 




A LABORATORY GUIDE IN HISTOLOGY. 



55 



tions ? Study the various stages of development between the primi- 
tive follicle and the fully developed follicle. Do any follicles have 
more than one egg-cell? Do you find a corpus luteum composed of 
relatively large cells having small nuclei ? If so, study carefully. 
See Fig. 50. Make a sketch under low power so as to show the 
general features of the ovary and sketch several follicles under 
the high power to show the stages of development. See Fig. 51. 

Ovary of a Very Young Animal. The ovary of a young 
kitten was treated as above. Stain in hematoxylin and eosin and 
mount. This is designed to show the development of the egg-tubes 
from the germinal epithelium. 

w\oxb\o\ ov>c\. 




*wb\vov\ oV vv^As oV 



OW\u. 



'"VAooo'VtmV 



Yvq3*. 



v3\ia\\j^ 0\tt \)tXU \XDWY\CV CVAO'. 



-~Y\^\ ot Ct\\% 



V1VXVY\, 



Study under low and high power. Find a downward growth 
of cells forming the egg-tube. Note that it is simply a downward 
proliferation of the cells from the layer of germinal epithelium into 
the stroma. Are all of the cells of the tube alike ? Study other 
tubes and note that certain cells form the ova and others form the 
follicular epithelium surrounding them. Search for a tube which has 
been penetrated by connective-tissue septa so as to divide it into 
somewhat distinct epithelial nests. The connective tissue later 



56 



A LABORATORY GUIDE IN HISTOLOGY. 



forms the theca folliculi surrounding the follicle. Sketch a portion 
of the section showing as many of these points as possible. See 
Fig. 5 2 - 







A\V&fo» 



V!W> t\AOw< \ vy>** . 



Fig. 53. Uterus of a Dog. 

Uterus. Portions of the uterus were fixed in a saturated 
solution of corrosive sublimate in normal salt solution, hardened in 
alcohol, imbedded in celloidin, and sectioned. Stain in haema- 
toxylin and Van Gieson's stain, dehydrate rapidly, clear in Eycle- 
shymer's mixture, and mount in balsam. 

Study under low and then under high power. How many 
layers do you find ? What kind of epithelium in the mucosa ? 
What is the nature of the tunica propria ? Are glands present in the 
mucosa ? Of what type are the glands ? What kind of muscle 
in the tunica muscularis ? How many layers ? What do you find 
between the layers? Study the vessels of the tunica muscularis. 
Study the tunica serosa which presents the usual characteristics of 
serous membranes. Sketch # a portion under low power. See Fig. 
53. Study and sketch as much of a gland as possible as seen under 
the high power. 



A LABORATORY GUIDE IN HISTOLOGY. 57 

Vagina. The vagina of a dog was fixed and hardened in 
alcohol, imbedded in celloidin, and sectioned. Stain in hsema- 
toxylin and eosin, and mount in balsam. 

Study under low power. What kind of epithelium do you 
find ? What are the characteristics of the tunica propria ? Do you 
find any adenoid tissue in the mucosa ? Are glands present ? Do 
lymph nodules occur ? Is a tela submucosa present ? Study the 
tunica muscularis. Does it have distinct layers ? Study the outer 
fibrous coat. Sketch as seen under low power so as to show the 
general structural features. Study under high power. 

Mammary Glands. 

Portions of mammary glands were hardened in alcohol, imbed- 
ded in celloidin, and sectioned. Stain in hematoxylin and eosin, 
dehydrate, clear, and mount. 

Study under low power. The lobules of the gland are com- 
posed of acini united by fibrous tissue. Search for and study the 
ducts. Study under high power. What variety of epithelium in 
the lining of the acini ? What is the position of the nuclei ? 
What do you find in the lumen of an acinus ? How do you account 
for the presence of fragments of epithelial cells ? Study the epi- 
thelium carefully and then sketch a portion of the gland as seen 
under high power. 

The Skin and its Appendages. 

Pieces of human skin were fixed in alcohol, and imbedded in 
paraffin. Fix the section to a slide, remove the paraffin and stain, 
one in haemotoxylin and eosin, and one in hematoxylin and Van 
Gieson's stain, clear in oil of bergamot and xylol, and mount in 
balsam. 

Study under low and high power. The layers of the epidermis 
are as follows, beginning with the outer one : ( i ) Stratum cor- 
neum or the horny layer. Are the cells well defined? Do they 
possess nuclei ? (2) Stratum lucidum. Why so called ? Of what 
form of cells is it composed? Is this layer granular? (3) Stratum 
granulosum. What is the form of cells? What is the nature of 
the granules in the cells of this layer ? (4) A layer of stratified 
squamous epithelium the deepest cells of which are columnar in 



58 



A LABORATORY GUIDE IN HISTOLOGY. 



form. Note the transition in form of the cells as you go to- 
ward the surface. Why does the form of the cell change? 

Study the dermis 
noting its general 
structure. Can you 
distinguish the pa- 
pillary and reticu- 
lar layers ? What 
is the general di- 
rection of the fibers 
in the pars retic- 
ularis? Note that 
the fibers of the 
pars papillaris are 
somewhat finer and 
hence the tissue is 
somewhat more 
dense than in the 
pars reticularis. 
Study the papillae. 
Study the glands of 
the skin under low 
and high power. 
Note their location 
and relations. 
Study the subcutaneous tissue. Draw and label a portion of the 
section as seen under the low power so as to show general relations. 
Sketch a few tubules of the sweat glands as seen under high power. 
See Fig. 54. 

The Scalp. Portions of human scalp were hardened in alco- 
hol, imbedded in celloidin, and sectioned transversely. Stain in 
hematoxylin and eosin, clear in Eycleshymer's mixture, and mount 
in balsam. 

Study under low power and then under high power paying 
especial attention to the hair and follicles and to the sebaceous 
glands. Sketch a hair and follicle as seen under high power, and 
label all the parts. See Fig. 55. Make a sketch of a sebaceous 
gland as seen under low power and show its relation to the hair 
follicle. 




Fig. 54. Transverse Section of Skin from the Human 
Heel showing the great development of the stratum 
corneum ; a, stratum lucidum ; b, stratum Malpighii. 



A LABORATORY GUIDE IN HISTOLOGY. 59 







\\o^V / w.otAfc wt X^o^vi. 









£ 






■'■ ■ ^^^y2^ T "^ :%: ~ * 



. •- './- v=^ wt\>^o Ov v\ oArv co 



-- r>-/ s 






V\0^4$>. S^ o^\W^o*W 



Foetal Scalp. Portions of foetal scalp were hardened in for- 
malin, imbedded in celloidin, and sectioned transversely. Stain in 
haematoxylin and Van Gieson's stain, dehydrate rapidty, clear in 



60 



A LABORATORY GUIDE IN HISTOLOGY. 



Kycleshymer's mixture, and mount in balsam. Study carefully, 
noting the development of the hair glands, and the relation of the 
glands to the other structures of the scalp. Sketch a portion as 
seen under low power. See Fig. 56. 

Scalp Showing Hairs in Transverse Section. Portions of 
scalp were imbedded in celloidin and sectioned tangentially. Study 
the follicles under high power and note the layers. Note the divis- 
ion into cortical and medullary portions. Study each carefully. 




\\Q^\\h>. iX\WfcO* ^\(KW lWoV \W\t ^00. 



Study the layers of the root sheath. The inner sheath has three 
concentric layers of cells ; the inner cuticle surrounding the hairs, 
the middle layer of Huxley composed of two layers of cells, and 
the outer single layer of clear cells or layer of Henle. The outer 
root-sheath has in its inner portion several layers of prickle cells 
composing the stratum Malpighii, the outer layer of which is col- 
umnar in form. Next in order is the glassy membrane, then the 
circular connective tissue, and then comes the looser outer connect- 
ive tissue with the bundles longitudinally placed. Sketch and label 
as seen under high power. See Fig. 57. 

Development and Structure of the Nail. The thumb of a 
foetus was hardened in formalin, imbedded in celloidin, and sec- 
tioned longitudinally. Stain in hematoxylin and eosin, clear in 



A LABORATORY GUIDE IN HISTOLOGY. 61 



oil of bergamot, and mount in balsam. Study the section under 
low power. See Fig. 58. Sketch. Study under high power re- 
membering that the nail itself represents an enormously developed 



y^ 





Fig. 58. L. S. Thumb of a Foetus x 30 : a., ossifying bone of 
the terminal phalanx ; b, hyaline cartilage ; c, blood-vessel ; 
d, stratum Malpighii ; e, matrix of nail. 

stratum lucidum. Study the cells of the nail. The nail bed is 
composed of the stratum Malpighii of the skin and the corium. 
The connective tissue fibers are somewhat coarser than they are in 
the corium of the skin. Study the matrix, the posterior portion of 
the nail-bed. 

Larynx, Trachea, and Lung. 

Larynx. The larynx of a rabbit was fixed and hardened in 
formalin, imbedded in celloidin, and sectioned transversely. Stain 
in hematoxylin and eosin, and mount in balsam. 

Study under the low power. What kind of epithelium do you 
find in the tunica mucosa ? Are goblet cells present ? Study the 



62 



A LABORATORY GUIDE IN HISTOLOGY 



tunica or lamina propria. Are papillae present ? Do you find any 
diffuse adenoid tissue ? Stud}' the tela submucosa. Look for mu- 
cous glands. Study the cartilages. What variety of cartilage is 
found in the larynx ? Study the perichondrium. Study the distri- 
bution of the blood vessels. Sketch a portion as seen under low 
power naming the parts. 

Trachea. The trachea of a cat was fixed and hardened in 
alcohol and imbedded in paraffin. Fix a section to each of two 
slides, remove the paraffin and stain, one in haematoxylin and eosin, 
and the other in Weigert's Fuchsin-resorcin for about half an hour, 
wash in alcohol, treat with absolute alcohol, clear with xylol, and 
mount in balsam. The elastic fibers should be stained dark blue 

and the nuclei should stain 
but little if at all. 

Stud}- under low and 
high power. The first 
preparation shows the gen- 
eral structure very nicely 
while the second shows the 
distribution of the elastic 
tissue. What is the gen- 
eral direction of the elastic 
fibers of the lamina pro- 
pria ? What type of glands 
do you find and where are 
they located ? Make a 
sketch under low power, 
using both sections, so as 
to show the structure of the 
trachea. See Fig. 59. 




Fig. 59. Trachea : a., epithelium : b. tunica 
propria; c. tela submucosa : d, elands; e, 
perichondrium. 



Lung, (a) Portions of the lung of a cat were fixed in 10% 
formalin, imbedded in celloidin, and sectioned. The lung of a 
larger animal is better for study and the portions should be kept 
entirely submerged in the fluid while undergoing fixation. Stain 
in haematoxylin and eosin, dehydrate, clear in oil of bergamot, and 
mount. Study under low power noting the sections of bronchi, 
blood-vessels and the lung tissue which is as seen in a collapsed 
lung. Compare your section with Fig. 60 and identify the parts. 



A LABORATORY GUIDE IN HISTOLOGY. 



63 



Study one of the larger bronchioles. What is the nature of the 
epithelial lining ? Are goblet cells present ? Do you find any 
glands in the mucosa ? If so, what kind ? What is the next tissue 




Fig. 60. Lung of a Cat: a., bronchiole; b, blood-vessel; c, 
respiratory bronchiole : d, lung tissue. 



beneath the mucosa ? Do you find any cartilage ? Does it occur 
as more or less complete rings or as plates somewhat irregularly 
disposed ? Do you find an outer fibrous covering? Do you find 
branches of the pulmonary artery in relation to the bronchiole ? 
Do you find a branch of the bronchial artery? Sketch the bronchiole 
and a little of the surrounding lung tissue as seen under low power. 

Study a small bronchiole as above. How does it differ in 
structure from a larger bronchiole ? Sketch as above. Study Fig. 
6 1 to learn the general relation of the blood-vessels to the lung 
tissue and then find a respiratory bronchiole and study carefully. 
Look for the capillaries often containing blood corpuscles. Study 
the epithelium of the air-cells. Are all of the cells alike? How 



64 



A LABORATORY GUIDE IX HISTOLOGY 



do the} 7 differ? Sketch a respiratory bronchiole and a portion of the 
epithelium of the air-cells as seen under high power. 

(b) The lung of a dog was distended with a 10^ solution of 
formalin after which the thorax was opened, the lungs removed and 



&\\-<o<l\ 




suspended in 10% formalin for a few days, after which portions 
were dehydrated, imbedded in celloidin, and sectioned. Stain and 
study as above noting the appearance of the distended lung and 



— CnWcMAI. 



-0AX-SC\C 










A LABORATORY GUIDE IN HISTOLOGY. 



65 



especially the epithelium of the air-cells. Sketch a portion of the 
lung tissue to show the epithelium and compare with the above. 

(c) Injected lung. The lung of a cat was injected with car- 
mine-gelatin through the pulmonary artery, hardened in alcohol, 
imbedded in celloidin, and sectioned. Stain in hematoxylin and 
eosin and mount. Study and then make a sketch of a portion to 
show the distribution of the pulmonary artery. See Fig. 62. 

(d) Elastic tissue of the lung. Lung tissue was hardened in 
alcohol and imbedded in paraffin. Fix a section to the slide and 
stain as follows : Place sections for twenty-four hours in orcein, 
(1-10 gm. of orcein in 20 c. c. of 95% alcohol, and 5 c. c. of dis- 
tilled water, to which a solution is added consisting of 20 c. c. 95% 
alcohol, 1 -10 c. c. hydrochloric acid, and 5 c. c. distilled water), 
differentiate in acid-alcohol for fifty or sixty seconds. Rinse in 
alcohol, and stain the nuclei with an alcoholic solution of methylene 
blue, rinse in alcohol, dehydrate in absolute alcohol, clear in xylol, 
and mount in balsam. Nuclei stain blue and elastic fibers a deep 
brown. Study carefully and sketch a portion to show the distri- 
bution of the elastic fibers. 

The Thyroid Gland. 



Portions of the thyroid of a dog were fixed in bichloride of 
mercury, hardened in alcohol, imbedded in celloidin, and sectioned. 
Stain in haematoxylin and eosin. 

Another portion of the gland was fixed in Flemming's solution 

and imbedded in 
JvAiAoWw\\«*«,. paraffin. Fix a 
section to the slide 
remove the paraffin 
with turpentine, 
and add xylol and 
the grades of alco- 
hol down to water, 
and then place 
them in 2.5% aque- 
o u s solution of 
ammonium sul- 
phate of iron for 







VuubV \\w<o\b G\cvv\C o)c"Doa* u \ 



00. 



66 



A LABORATORY GUIDE IN HISTOLOGY. 



about five hours. Rinse in water and stain in the following solu- 
tion of hsematoxylin : Hematoxylin crystals, i gm. ; absolute 
alcohol 10 c. c; distilled water 90 c. c. ; diluted when used with an 
equal volume of distilled water. They should be stained for 
twenty-four hours, rinsed in water, and again placed in ammonium 
sulphate of iron solution until the excess of stain is removed, after 
which they are washed carefully in distilled water, dehydrated, 
cleared in xylol, and mounted in balsam. This is known as 
Heidenhain's Iron-Hsematoxylin Stain. 

Study the first section under low power. Note the fibrous 
capsule investing the gland. What is the shape of the alveoli? 
How are they lined? Study the colloidal substance. Does it stain 
uniformly and what is its composition. Study the second section as 
above, compare with the first, and then sketch a few alveoli to show 
their appearance under the high power. See Fig. 63. 

Huber recommends fixing with Flemming's fluid and staining 
with the Ehrlich-Biondi mixture. This stain serves to differentiate 
the chief from the colloidal cells. The chief cells do not stain. 
while the colloidal cells are red with green nuclei. This method 
has given good results with us. 

The Eye. 

Cornea. The eye of a man was removed, cleaned of superfluous 
fat and connective tissue, placed in Flemming's solution and divided 
into anterior and poster- 



ior halves. After fixing 
the lens was removed 
and one fourth of the 
anterior half was stained 
in hematoxylin, dehy- 
drated, cleared, and im- 
bedded in paraffin. Fix 
your section to the slide, 
remove the paraffin with 
xylol, and mount in bal- 
sam. 

Study the cornea 
noting carefully the fol- 
lowing layers : (1) 




COYY\ec\ 



OYO^YVC^. 



53-- tOV* 



l(A tOYQvAVM 



tS. 



V'va.W. oAuwKftw Vovwta \ MSo. 



Iw o pvrv»\ * v> *** ■ 



A LABORATORY GUIDE IN HISTOLOGY. 



67 



The corneal epithelium. How many layers of cells in it and of what 
variety are they? (2) The second layer is the anterior elastic mem- 
brane. What can you determine as to its structure? Does it extend to 
the sclera? (3) The third layer is the substantia propria or corneal 
ground-substance. Study the structure carefully noting that it 
seems to be composed of fibrous tissue, both in bundles and 
lamellae, between which cells or corneal corpuscles are found in the 
corneal spaces. Is the substantia propria continuous with the con- 
nective tissue of the sclera ? (4) The posterior elastic membrane. 
What is its structure ? (5) The endothelium of the anterior cham- 
ber. Sketch a portion as seen under high power. See Fig. 64. 

The Sclera. Study carefully, especially near and at its junc- 
tion with the cornea. Of what layers is it composed? Study 
the canal of Schlemm. Study also the relations of the iris and 
ciliary processes as shown in the section. Sketch to show the 
general structural relations. 

The Choroid Coat. Portions of the posterior half of the eye- 
ball were imbedded in paraffin, and sectioned transversely. Fasten 
to the slide, remove the paraffin, stain in haematoxylin and eosin, 
and mount in balsam. Study carefully under low and high power 
and try and distinguish the following layers from without inward : 
(1) The lamina suprachoroidea. What is its composition? To 
what layer of the sclera is it joined ? Are pigment cells present ? 
Do you find the perichoroidal lymph-spaces ? How are they formed 




--u\a»>*u\{\\xt*. 



L& oViovkocaoiuuvt*. 






** ft, 



\vt\AoV i\ww\UACWovovOk X\00. 



and with what are 
they lined? (2) 
The lamina vascu- 
losi. What is its 
structure? How 
does it differ from 
the above in struc- 
ture ? How are the 
bloodvessels dis- 
tributed ? Locate 
and study the 
boundary zone. (3) 
Lamina choriocap- 
illaris. What is its 



68 



A LABORATORY GUIDE IN HISTOLOGY 



structure? Are pigment-cells present? (4) The vitreous membrane. 
What is its general structure ? Sketch a portion to show these 
layers and their relative thicknesses and relations. See Fig. 65. 

Nervous Tunic. Stud}* the preparation used above. Note 
that this coat has two layers, (1) the pigment layer and, (2) the 
retina. What is the shape of the cells in the pigment layer? What 

► ., v is the position of 




.x%V\W<*. 







\\^Vv^A ; 



the nuclei ? Study 
the relation of the 
processes of the 
cells. The section 
of the retina lies be- 
tween the ora ser- 
rata and the macula 
lutea and the fol- 
lowing layers 
should be discerned 
and carefully stud- 
ied from without 
inward : (1) The 

layer of visual cells 
which consists of 
cone- visual cells 
and rod-visual cells. 
Try and distin- 
guish them and 
study their relation 
Of what elements does 
What is its structure ? 



carefully. (2) The outer molecular layer. 

it consist? (3) The inner nuclear la5'er. 

(4) The inner molecular layer. What is its formation ? (5) The 

laj-er of ganglion cells. What type of ganglion cell do you find ? 

Study the cell processes. (6) The nerve fiber layer. Are the 

fibers medullated or non-medullated ? Make a sketch showing 

these la3 T ers and their component parts. See Fig. 66. 

The Crystalline Lens. Macerate the lens for twenty-four 
hours in Ranvier's one-third alcohol, tease on a slide, cover and 
study. What is the shape of the fibers ? Are they nucleated ? 
How many nuclei ? Sketch a few fibers as seen under high power. 



A LABORATORY GUIDE IN HISTOLOGY. 69 



The Eyelid. The eyelid was fixed and hardened in alcohol, 
and imbedded in paraffin. Fix a section to the slide, remove the 
paraffin, stain in haematoxylin and eosin, and mount. Study under 
low and high power noting the three layers: (i) The cuticular 
layer with a very thin epidermis. Do you find dermal papillae ? 
Are they of uniform size and distribution ? What variety of glands 
do you find ? Do you find any pigment-cells ? (2) The middle 
layer. What kind of cartilage do you find ? What kind of muscle 
do you find? Note the distribution of bloodvessels. What kind 
of connective-tissue do you find ? Has it any definite arrange- 
ment ? (3) The conjunctiva. What kind of epithelium do you 
find ? Are goblet-cells present ? Is lymphoid tissue present ? 
Is it diffuse or nodular ? Do you find glands ? Sketch a portion 
of the section to show the structural relation as seen under low 
power. 

The Ear. 

External Ear. Portions of the external ear were fixed and 
hardened in alcohol, imbedded in celloidin, and sectioned. Stain 
deeply in the elastic tissue stain and mount. Study under low 
power noting the general features of the section. The skin is 
rather thin in the external ear. Do you find hair-follicles ? Do 
you find sebaceous glands ? Are the sweat-glands present on both 
surfaces? What kind of cartilage do you find? Are the elastic 
fibers uniformly distributed ? Sketch under low power to show the 
general structural features. 

Inner Ear. The cochlea of a guinea-pig was fixed for twelve 
hours in Flemming's solution, and after washing, was decalcified in 
2% hydrochloric acid (2% nitric acid, or 1 % chromic acid would 
do), imbedded in celloidin, sectioned parallel to the longer axis of 
the section, and stored in 80% alcohol. Stain in haematoxylin and 
eosin, and mount in balsam. 

Study the section carefully under low power. Do you find the 
axis of the cochlear canal ? Do you find the lamina spiralis ossea 
which separates the canal into the scala vestibuli and the scala 
tympani ? How are these portions of the canal lined ? Can you 
find the cochlear duct ? If so try and find the organ of Corti 
and identify and study its parts. The pillars of Corti form a sort 



70 A LABORATORY GUIDE IN HISTOLOGY. 



of arch. Do you find the hair-cells ? Look for the lamina reticu- 
laris and the membrana tectoria. Try and find the cells of Deiter 



\©YV. 










X\tX\>t 



H 



sN> 



VVfe. 



and those of Hensen. Sketch as seen under low power and identify 
and label all of the parts by comparing with the cuts or with the 
aid of the instructor. See Fig. 67. 

Foetal Ear. Stain the sections of human foetal ear in hsema- 
toxylin and eosin, and mount. Study carefully and compare with 
the section given above. Sketch and label the important parts. 

The Olfactory Mucous Membrane. 

Isolated Epithelium. Portions of the mucous membrane of a 
dog's nose were macerated in Ranvier's one-third alcohol for two 
hours and were then placed in a 1% solution of osmic acid for 
twenty minutes. Tease carefully on a slide and examine. Stain 
another portion with methylene blue, wash, tease, and mount in 
Farrant's gum glycerin. 

Study under high power. What forms of epithelial cells do 



A LABORATORY GUIDE IN HISTOLOGY. 



71 



- V\,VXtA.4.\AS. 






you find ? How can you distinguish between the olfactory cells 
and the sustentacular cells ? Sketch and label as seen under high 
power. 

Epithelium in Sec- 
tion. Portions of the 
olfactory mucous 
membrane were fixed 
and hardened in alco- 
hol, imbedded in cel- 
loidin, and sectioned. 
Stain in hematoxylin 
and eosin, clear, and 
mount. Study the 
epithelium, carefully 
noting its structure. 
Do you find a base- ' . v \Wb^>w*\. 

ment membrane ? Do \ \cA>V CAW^o\'\A\Y\uto^XKVfcv^Y<xv\t oW^ovn- 
you find glands ? Are 

they of the serous or mucous type ? How are the blood vessels 
distributed ? Sketch to show the most important structures. See 
Fig 68. 




Nerve Histology. 



Nerve Cells. 



\ 



\ *t©V\. \WY\jt-ctW*, S^\xio\(/wo ox. Ox:. 



With a scalpel remove a small portion of the 

gray matter 
from the ante- 
rior horn of the 
spinal cord of 
the ox, place it 
between two 
clean cover- 
glasses and 
make a cover- 
glass preparation 
or smear. Allow 
the smear to dry 
in the air and 
then place it in 
cover-glass for- 







72 A LABORATORY GUIDE IN HISTOLOGY. 

ceps and pass quickly two or three times through the flame of a 
Bunsen burner. Stain for twenty or thirty minutes in a i % aque- 
ous solution of methylene blue. Wash in water, dry thoroughly 
between pieces of filter paper, or in the air, and mount in balsam. 

Study the preparation under low and then under high power. 
Note the general form of the cell-body. Are the cells unipolar, 
bipolar, or multipolar? Do you find a neuraxis? Are the cells 
alike as to form and size ? Is the protoplasm clear or granular ? 
Can you see any fibrillation in the protoplasm under the high 
power ? Are nucleoli present ? Can you find the implantation 
zone? Make drawings. See Fig. 69. 

Nerve Cells Macerated. Macerate portions of the gray matter 
from the anterior horns of the spinal cord for a week or ten days in 
Ys alcohol, a 1 % solution of potassium dichromate, or a 5% solution 
of chromic acid. Thionin is a good stain to use after alcohol as a 
fixative. Lithium carmine after chromic acid is recommended by 
Huber. The stained material should be carefully teased and ex- 
amined in glycerin, or it may be mounted in gum glycerin. Study 
and note the structure and form as above. You should be able to 
trace portions of the neuraxes. Sketch a cell showing the neu- 
raxis. 

Nerve Cells of the Cerebral Cortex. Small pieces of the cere- 
bral cortex of a cat were placed in the following mixture, as recom- 
mended by Fish : 

Formalin 2 c. c. 

Potassium dichromate, 3%, 100 c. c. 
for three days, using fifty times their volume of fluid, and kept in 
the dark. The tissue was then placed in the 3% potassium dichro- 
mate solution for three or four days. The potassium dichromate 
was replaced by a ^ % solution of silver nitrate until the chromate 
of silver was no longer precipitated. A fresh solution of silver 
nitrate was poured over the tissue and it was kept in the dark for 
from three to five days. It is best to change the silver nitrate solu- 
tion after ten to twelve hours. The tissue was then imbedded in 
celloidin as follows: Dehydrate in 95% alcohol, 15-20 minutes. 
Change the alcohol once or twice and agitate. Absolute alcohol 
for 30-40 minutes. Ether-alcohol, 20 minutes. Ether, 15 minutes. 
Thin celloidin, 30 minutes. Thick celloidin, 15 minutes. Tissue 
was mounted on blocks and placed in chloroform for 10 minutes, 



A LABORATORY GUIDE IN HISTOLOGY. 



73 



tfc\\-V>00i 



iX \ 



vXt,- 






after which it was placed in a modified Eycleshymer's clearing fluid 
(carbolic acid, 25 parts; oil of cedar, 50 parts; and oil of bergamot, 
50 parts), for one hour. The tissue may remain in oil a day or 
two without seeming to suffer much injury. Sections were cut with 
a sliding microtome, using the clearing fluid to wet the tissue and 
the knife, and transferred to a dish of clearing oil. Take a section 
from the clearing oil and place it on a clean slide, press down and 
blot with a piece of filter paper, and cover with a large drop of very 
thick Canada balsam. Do not use a cover-glass and dry quickly in 
a warm, dry place protected from dust. 

Study the section with 
low power. The plexus of 
blood vessels may confuse 
you but search for py- 
ramidal cells, stained a 
brownish-black, with a 
single neuraxis extending 
from the base of the cell- 
body into the white matter, 
and branching dendritic 
processes extending toward 

the surface of the cortex V\^.Ho.^vc\m\kc\ Ci\\% oVoC*\. &oW 
Only portions can be found ° 

in connection with a single cell. Note the size of the dendrites as 
compared with the neuraxis. Do the cells have a definite arrange- 
ment? Could they be said to have a characteristic form ? Do they 
have more than one dendrite? Note the branching of the dendrite. 
Sketch several pyramidal cells and label the parts. See Fig. 70. 

Nerves. 

Fresh Fibers. Carefully remove the sciatic nerve from a frog 
and place it in physiological normal salt solution (.6% ). With very 
sharp scissors remove a piece about one-half inch in length and 
place it on a slide and tease carefully keeping the fibers as nearly 
straight as possible; cover with a cover-glass and examine first with 
low power, and then with the high power. Use a small diaphragm 
opening for the high power. 

Search for the neuraxis or axis cylinder seen as a light band 
running down through che center of the fiber. Outside of the 




tW«\X\«>. 



74 



A LABORATORY GUIDE IN HISTOLOGY. 



neuraxis is the myelin sheath which has a glistening appearance 
with a slightly greenish tint. Do 3^011 find the nodes of Ranvier ? 
Estimate the length of the internodal segments. Look for the 
neurilemma as it crosses from one internode to another. Is the 
medullary sheath continuous from internode to internode ? Do you 
find the nuclei of the internodal segments ? Look for the segments 
of Lantermann. Do you find the long, oval neurilemma-nuclei ? 
Note the contraction ring of the neurilemma at the nodes. Are all 
the fibers of uniform diameter ? Do they branch ? Make a sketch 
to show as many of these points as possible. 

Stained Nerve Fibers. The sciatic nerve of a frog, or a 
small nerve of a mammal, was placed in a .5% solution of osmic 
v acid for twenty-four hours, 



r.- 






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washed in water, and stored in 
70% alcohol. Take the portion 
given you and tease carefully on 
a slide in dilute glycerin, cover 
and examine, first with the low 
and then with high power. The 
medullar}- sheath is stained a 
deep black. Why ? Can you 
trace the neuraxis? Study the 
nodes and the neurilemma con- 
traction ring. Do you find the 
segments of Lantermann? Sketch 
a node of Ranvier with portions 
of the internodal segments as 
seen under high power. See 
Fig. 71. 

Stained Nerve. A section 
of human ulnar nerve was hard- 
ened in formalin, imbedded in paraffin, and sectioned longitudinally. 
Fasten to a slide with albumin fixative and stain in haematoxylin, 
Boehmer's is best. Study under high power. What is the appear- 
ance of the medullary sheath ? Are the internodal nuclei visible? 
The neuraxis usually stains deeply. Sketch a portion of the section. 

Stained Nerve. A cross section of the same nerve was hard- 
ened in Miiller's fluid, inbedded in celloidin, and sectioned. Stain 




A-W\^A\u\toVvW. 

V\u*n. XWvx. vCfctv *. 



A LABORATORY GUIDE IN HISTOLOGY. 



75 



in aniline blue and safranin, dehydrate, clear in oil of bergamot, and 
mount in balsam. Study under low power noting the epineurium 




YVeiAV\VAW\. 



Y\?AAYC\\W 
^Y\Y\tWX\UVV\. 



Y\<\.1 a.^.S.^v 



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V(W. 



and the perineurium. The neuraxes are stained blue, the nuclei 
red, and medullary sheaths a reddish-yellow. Sketch a portion care- 
fully, labeling all of the parts. See Fig. 72. 

Non-meduIIated Nerves, (a) The splanchnic nerves of a dog 
were fixed in formalin, and stained in hematoxylin. Tease care- 
fully in glycerin and study under the high power. Are the fibers 
medullated or non-medullated ? Do the non-medullated fibers 
possess nodes of Ranvier? Do they have a primitive sheath or 
neurilemma ? Do you find nuclei ? Sketch a typical non-medul- 
lated fiber. See Fig. 71. 

(b) Pieces of the vagus nerve were treated with a .5-1% solu- 
tion of osmic acid for twenty four hours and they are to be care- 
fully teased and examined in dilute glycerin. Between the medul- 
lated fibers the non-medullated are found. Compare with (a) above 
and sketch a few fibers. 

Ganglia. Cross sections of the spinal cord of a cat were made 
so as to pass through the posterior root ganglia. Stain deeply in 
Ehrlich's acid-hsematoxylin and Van Gieson's stain. Dehydrate, 
clear in Eycleshymer's nerve mixture, and mount in balsam. Study 
under low power. Note the capsule of the ganglion and its septa 



76 



A LABORATORY GUIDE IN HISTOLOGY. 



and trabecule. How are the ganglion cells arranged ? Study the 
cells under high power. Are they unipolar or bipolar? Note 
the large nucleus and the nucleolus. What is the relation of the 



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\ \ OyJV*. b tA\<WS oV ^ 



^>(\i\Co; 



d\>3<xyvc^> 



cells to each other ? Do you find any fibers ? Do any fibers end 
in the ganglion ? Sketch under low power to show general relation 
and arrangement, and under the high power to show as much as 
you can of the ganglion cell and its structure. See Fig. 73. 

Nerve Endings. Treat pieces of the ocular or intercostal mus- 
cles, other small muscles would do as well, by Ranvier's lemon- 
juice method as follows : The lemon- juice should be filtered 
through clean new flannel, after which pieces of the tissue are 
placed in it until they become transparent, usually requiring five 
or ten minutes, after which they are to be quickly rinsed in distilled 
water and placed 



in the dark in a 
1 °/ solution of gold 
chloride for twenty 
or twenty -five min- 
utes, after which 
they are to be 
washed in water 
and placed in dis- 
tilled water to 




j.....AWJAC\ 



: -..w«.><»«.. 






\\^\.\^&o< K\twt.t*V\Yva.VWv&%Ve<W^t. 



A LABORATORY GUIDE IN HISTOLOGY. 77 

which acetic acid has been added in the proportion of one drop of 
acid to each 25 c. c. of water. Now expose to the light for at 
least twenty-four hours. The reduction of gold is not very com- 
plete and such material is only suited for present study. Permanent 
preparations may be made by placing the tissue in formic acid of 
25% strength. The reduction is usually complete in twenty-four 
hours. 

Tease the tissue very carefully on a slide and search for the 
motor-endings. Study carefully and make several sketches to show 
typical forms of endings. See Fig. 74. 

Crosses of Ranvier. — Silver Nitrate Method. Place portions 
of a small nerve, or spinal nerve roots, in a 1 % solution of silver 
nitrate. Keep it in the dark for about twenty-four hours. Remove 
from the silver solution, rinse quickly in distilled water and place 
on a slide in a drop of glycerin and carefully tease one end of the 
fiber, cover, and expose to the sunlight for twenty-five to thirty 
minutes. 

Study under high power. Do you find the crosses ? Why are 
crosses formed at the nodes ? Do you find the transverse striations 
known as Frommann's lines? Sketch. 

Spinal Cord. 

A portion of spinal cord from the cervical region of a cat was 
hardened in corrosive sublimate in normal salt solution, or in for- 
malin. After further treatment with iodized alcohol and pure alco- 
hol, in case the sublimate be used, the tissue was imbedded in cel- 
loidin, and sectioned. You will stain the section on a slide in borax 
carmine, dehydrate, clear in Kycleshymer's mixture, and mount in 
balsam. 

Study under the low power and note the form of the cord, fis- 
sures, membranes and nerve-roots. Examine the gray matter, not- 
ing the relative size and position of the anterior and posterior corim. 
Do you find any definite regions or cell areas in the gray matter ? 
Name them by consulting the figure. Examine the white matter 
more carefully, trying to see if it is divided into definite areas. 
Sketch the cross-section under the low power being careful to pre- 
serve the proper proportions and to keep the relative size of the 
various parts. See Fig. 75. 



78 



A LABORATORY GUIDE IN HISTOLOGY. 



A section of the spinal cord of an ox was hardened in fluid 
consisting of 3% potassium bichromate, 98 parts, and formalin, 2 
parts. The tissue was imbedded in celloidin and sectioned. Stain 
the section you receive in Weigert's haematoxylin. wash, dehydrate, 

AtoA. 







"\^t&*ncttift, o\S>o\^^o^. 



clear, a xylol-carbolic acid mixture is good, or in Eycleshymer's 
nerve fluid, mount in balsam, and study as above, noting the finer 
details in the structure under the high power. Do not attempt to 
make a complete sketch but fill in portions of both gray and white 
matter in a complete outline sketch of the cord. 

This portion was stained by the rapid Golgi method (mentioned 
on page 72), imbedded in celloidin, and sectioned. Remove the 
excess of clearing oil and mount in very thick balsam, without a 
cover-glass. Study under low power and make drawings of typical 
cells and structures. 



Spinal Cord Stained in Nigrosin and Eosin. Stain the section 
for one hour in a 1 ^ aqueous solution of nigrosin. Wash in distilled 
water for five minutes. Stain for five minutes in a 1 °] solution of 
eosin. Wash in 70% alcohol for several minutes and place in 95% 
alcohol until stain ceases to be given off. Clear in oil of bergamot 
for ten to fifteen minutes, and mount in balsam. Studv as above. 



80 



A LABORATORY GUIDE IN HISTOLOGY 



?--moi^ 



fcx\0*\a 



cells do you find, large or small ganglion cells? What tissue seems 
to form the greater part of this layer ? (2) Cell layer of Purkinje's 
cells. Can you trace an axon from the base of the cell-body ? 
How many dendritic processes at the opposite pole? How do they 
branch? Do the branches seem to have varicose enlargements? 
Do they anastomose? Do you find basket cells? (3) Granular 
layer containing small granular (ganglion) cells and large stellate 
cells. How many dendrites do the small cells have and how do 
they end ? Can you trace the neuraxes toward and into the 

molecular layer? Look for 
the large stellate cells close 
to the molecular layer, not- 
ing the branching and 
course of the dendrites and 
neuraxes. Sketch a por- 
tion showing as much as 
you can of the cortical 
layers. See Fig. 76. 

Golgi Method. Portions 
of the cerebellar cortex 
were treated by the rapid 
Golgi method mentioned 
on page 72. Mount as 
there described and study 
and compare with the 
above. Sketch as seen under low power, and sketch several cells 
of Purkinje as seen under high power. See Fig. 77. 

The Cerebrum. 

Stained Cortex. Small pieces of the cerebral cortex of a cat 
were fixed in bichloride of mercury, hardened in alcohol, imbedded 
in celloidin, and sectioned. Stain in haematoxylin, and, very 
lightly, in eosin, dehydrate, clear, and mount in balsam. This sec- 
tion is useful for the purpose of showing the general layers. What 
kind of tissue composes the larger portion of the molecular layer ? 
Do you find any nerve cells ? Look for a thin stratum of small 
medullated fibers just beneath the pia mater. Is the molecular 
layer sharply marked off from the layer beneath it? Describe the 










A LABORATORY GUIDE IN HISTOLOGY. 



81 




'yW\CViVv\txA^v\t<. 






$<n\to*\o*^\vou% u\\v 



structure of the granular layer. Note the relative number of cells 
in the granular and molecular layers. 

Golgi Cortex. Pieces of 
the cerebral cortex were 
prepared by the rapid 
Golgi method described on 
page 72, imbedded in cel- 
loidin, and sectioned. Re- 
move the excess of clearing 
fluid and mount in a drop 
of very thick balsam, with- 
out using a cover-glass. 
Study under low power, 
noting carefully the form 
and relative number of cells 
in each layer. How are the 
cells distributed in the 
outer layer ? Do they pos- 
sess typical forms? Do 

you find distinct bound- \'\aM UW t&xU.fcJw^^ 

aries between the layers? ° » \ \ 

Note the relative size of the cells of the pyramidal layers. From 
what portion of the cell body is the axon given off ? How many 
dendrites, and where do they terminate ? What forms of cells do 
you find in the layer beneath the layer of large pyramidal cells ? 
Make a sketch showing the structure of the cortex. See Fig. 78. 

Nerve Fibers of the Cortex. A portion of the cerebral cor- 
tex of a dog was fixed in Miiller's fluid, hardened in alcohol, im- 
bedded in celloidin and sectioned. Stain the sections in Weigert's 
haematoxylin, dehydrate, clear, carbol-xylol is best for clearing, 
and mount. The nerve fibers are stained a blue-black color. Note 
carefully the distribution of nerve-fibers in the cortex. Sketch a 
portion of the cortex, using the low power. 



82 A LABORATORY GUIDE IN HISTOLOGY. 

Formulas for Reagents. 

A. STAINS. 

(1) Boehmer's Haematoxylin. 

Hematoxylin crystals, i gram. 
Absolute alcohol, 10 c. c. 
Potassium alum, 10 grams. 
Distilled water, 200 c. c. 

Dissolve the alum in the water and the haematoxylin in the alco- 
hol. Mix while stirring constantly. Place in an open jar, protect from 
dust, and let ripen for two or three weeks. Filter, and the stain is 
ready to use. 

(2) Delafield's Haematoxylin. 

Haematoxylin crystals, 4 grams. 

Absolute alcohol, 25 c. c. 

Sat. aq. solution of ammonia alum, 400 c. c. 

95% alcohol, 100 c. c. 

Glycerin, 100 c. c. 

Dissolve the haematoxylin in the absolute alcohol and add to the 
solution of alum. Place in an open dish and expose to the light 
for four or five days and filter. Add the 95% alcohol and the 
glycerin and let the solution ripen for six or eight weeks. It is 
now ready for use after filtering. It is diluted with distilled water 
before using. 

(3) Ehrlich's Acid Hematoxylin. 

Haematoxylin crystals, 2 grams. 
Absolute alcohol, 100 c. c. 
Glacial acetic acid, 10 c. c. 
Glycerin, 100 c. c. 
Distilled water, 100 c. c. 
Potassium alum, in excess. 

This stain should ripen in the light for some time until it ac- 
quires a dark-red color. It 'stains and keeps well for years if kept 
in well-stoppered bottles. 



A LABORATORY GUIDE IN HISTOLOGY. 83 



(4) Aqueous Solution of Borax-Carmine. 

Grind 2 grams of carmine with 8 grams of borax in a mortar, 
and add it to 150 c. c. of distilled water. Let it stand for from 
twenty-four to thirty-six hours and filter when it is ready for use. 

(5) Safranin. 

Safranin, 1 gram. 

Aniline water, 90 c. c. 

Absolute alcohol, 10 c. c. 
Prepare the aniline oil by shaking 8 to 10 c. c. of aniline oil in 
100 c. c. of distilled water, and filtering through a wet filter. Dis- 
solve the safranin in the aniline water and then add the alcohol. 
It must be filtered before using. 

(6) Weigert's Hematoxylin. 

Hematoxylin crystals, 1 gram. 
Absolute alcohol, 10 c.c. 
Lithium carbonate, 1.2 grams. 
Distilled water, 100 c. c. 
Dissolve the hsematoxylin crystals in alcohol, the lithium car- 
bonate in water, and mix the two solutions. 

(7) Congo Red. 

Make a .5-1% aqueous solution. It is particularly useful in 
staining the parietal cells of the cardiac end of the stomach since 
it seems to have a special affinity for acid. It is also used as a stain 
for the axis-cylinder of a nerve fiber. 

(8) Thionin. 

A concentrated aqueous solution is usually used. It stains 
chromatin readily, and stains plasmatic elements if the staining 
process be prolonged. It is more used as a specific mucin stain. 

(9) Orcein, Israel's Formula. 

Orcein, 2 grams. 

Glacial acetic acid, 2 c. c. 

Distilled water, 100 c. c. 
This gives a blue stain in the nucleus and a red stain in the 
cytoplasm. After staining wash the section in distilled water, 
dehydrate rapidly, passing through absolute alcohol to thick cedar 
oil in which it is mounted. 



84 A LABORATORY GUIDE IN HISTOLOGY. 



(10) Van Gieson's Stain. 

(a) Make a saturated aqueous solution of acid fuchsin. 

(b) Make a saturated aqueous solution of picric acid. 
Add (b) to (a) until the mixture is garnet red. 

(11) Elastic Tissue Stain of H. G. Harris. 

Hematoxylin, 0.2 gram. 
Aluminium chloride, o. 1 gram. 
50% alcohol, 100 c. c. 

(12) Acid Fuchsin. 

Use a 0.5- 1 % aqueous solution. 

(13) The Ehrlich=Biondi Mixture. 

Take 200 c.c. of a saturated aqueous solution of orange G and 
add to it while stirring 40 c.c. of a saturated aqueous solution of 
acid fuchsin, and 100 c.c. of a saturated aqueous solution of methyl 
green. The saturated solutions should be prepared several days 
before using and they must be completely saturated. Sections 
should be stained for from twelve to twenty-four hours, dehydrated, 
cleared with xylol, and mounted in xylol-balsam. 

(14) Heidenhain's Iron=Haematoxylin Stain. 

Hematoxylin crystals, 1 gram. 

Absolute alcohol, 10 c. c. 

Distilled water, 90 c.c. 
To be diluted with an equal volume of distilled water when 
used. A mordant of a 2.5% solution of ammonium sulphate of 
iron is used for five or six hours. After staining for twenty-four 
hours, the ammonium sulphate of iron solution is used to remove 
the excess of stain. 

(15) Eosin. 

Eosin is commonly used as a 2% solution in 60% alcohol, 
though saturated aqueous solutions may be used as well as satu- 
rated alcoholic solutions. 

B. MACERATING FLUIDS. 

(l) Ranvier's One=third Alcohol. 

Fresh tissue should be macerated from eighteen to twenty-four 
hou^s, after which it may be teased on a slide in a drop of dilute 



A LABORATORY GUIDE IN HISTOLOGY. 85 

glycerin. Or, after macerating, treat for several hours in a 5% 
solution of osmic acid before teasing. 

(2) Hydrochloric Acid. 

Hydrochloric acid in a 25-30% solution is commonly used to 
macerate kidney tissue. It should act at least twelve hours after 
which the tissue is washed and teased in water or dilute glycerin. 

(3) Caustic Potash. 

A 30% aqueous solution is much used for macerating cardiac 
and smooth muscle. It should act for about twenty minutes after 
which it is placed in a saturated aqueous solution of potassium 
acetate to which a little glacial acetic acid has been added in the 
proportion of about one drop of acid to 5 c.c. of the acetate solu- 
tion. After twenty-five minutes the tissue may be teased and 
examined. 

(4) Nitric Acid. 

It may be used in a 30% .solution, or in a 20% solution of fum- 
ing nitric acid. 

(5) Chromic Acid. 

This is used in aqueous solutions of from 0.2-1% strength. It 
is used to macerate smooth muscle, and may be used for nerve tis- 
sues. 

C. FIXING FLUIDS. 

(1) Bichloride of Mercury. 

A saturated solution in distilled water is used. A saturated 
solution in physiological normal salt solution (.6%) makes a good 
fixing agent. 

Huber combines it with formalin. This seems to be useful in 
embryonic tissue, especially developing bone. He adds four parts 
of formalin to one hundred parts of a saturated aqueous solution of 
the bichloride of mercury. 

(2) Miiller's Fluid. 

Bichromate of potassium, 2.5 grams. 
Sulphate of sodium, 1 gram. 
Distilled water, 100 c.c. 
This solution is much used for nerve tissues and for large pieces 
of tissue. One objection to its use is that it acts very slowly, and 



86 A LABORATORY GUIDE IN HISTOLOGY. 

hence it should not be used if the finer details of cell structure are 
desired, as post mortem changes usually occur to some extent be- 
fore the killing and fixing process begins. 

(3) Flemming's Solution. 

2% aqueous solution of osmic acid, 4 c.c. 
1% aqueous solution of chromic acid, 15 c.c. 
Glacial acetic acid, 1 c.c. 

It is best to keep the osmic acid and chromic acid in separate 
bottles and combine them and add the acetic acid when wanted for 
use. 

(4) Carnoy's Fluid. 

2 or 3% aqueous solution of chromic acid, 45 c.c. 
2% aqueous solution of osmic acid, 16 c.c. 
Glacial acetic acid, 3 c.c. 

(5) Alcohol. 

Small pieces of tissue may be placed in absolute alcohol, being 
killed, fixed, hardened, and dehydrated at the same time. Unless 
absolute alcohol is used, 35% alcohol should be used for a few 
hours, followed by 50%, 70%, 85%, etc., as tissue placed directly 
into 95% alcohol suffers considerable shrinkage. 

(6) Formalin. 

Formalin in a 5-10% solutionis much used, especially for nerve 
tissues. It is not best to use it in case white fibrous connective tis- 
sue predominates in the section as it hardens it very greatly. Nor 
is it a good fixing agent for portions of such tissues as the stomach 
as it does not harden the tissue uniformly and it does not cut well. 

(7) Chrom-acetic Fluid. 

Chromic acid, 2 grams. 
Glacial acetic acid, 2 c.c. 
Water, 296 c.c. 

Mayer's Albumin Fixative. 

This is made by taking equal parts of white of egg and glycerin 
and adding to it a little salicylate of soda or gum camphor to pre- 
vent decomposition. 



A LABORATORY GUIDE IN HISTOLOGY. 87 



Acid Alcohol. 

Add to 50% or 60% alcohol about 2% of hydrochloric acid. 

Ammonia Alum. 

For use in removing the excess of hematoxylin after over- 
staining take a 2-3% aqueous solution of ammonia alum. 

Lugol's Solution. 

Distilled water, 100 c.c. 
Potassium iodide, 6 grams. 
Iodine, 4 grams. 

Carmine-gelatin Injection Mass. (Ranvier.) 

Soak fifty grams of Paris gelatin in water for thirty minutes, or 
until it becomes soft, and then melt it in the water it has absorbed 
in a porcelain vessel over a water-bath. When it is completely 
melted add a solution of five grams of carmine in just enough 
ammonium hydrate to effect the solution of the carmine forming a 
transparent solution. Stir the mixture constantly keeping it warm 
over the water-bath. The excess of ammonia must be neutralized. 
Add drop by drop, while stirring, a solution of one part of glacial 
acetic acid to three parts water. When you are near the point of 
neutrality dilute the acid still further. The smell gradually changes 
from ammoniacal to the sour odor of the acid. This must be done 
slowly and carefully as a single drop of acid in excess may spoil the 
mixture. Filter through clean flannel. 

Farrant's Gum-glycerin. 

Glycerin, 50 c.c. 
Distilled water, 50 c.c. 
Pure gum-arabic, 50 grams. 
Arsenious acid, 1 gram. 
Dissolve the acid in water. Mix with the gum-arabic in a 
mortar. Add the glycerin and filter through fine new muslin. 

Stock or Thick Solution of Celloidin. 

Dissolve 12 grams Schering's celloidin in 200 c.c. of equal parts 
of absolute alcohol and ether. A thin solution is prepared by tak- 
ing equal parts of the stock solution and the absolute alcohol and 
ether solution. 



88 A LABORATORY GUIDE IN HISTOLOGY. 

D. CLEARING FLUIDS. 

Purposes of a clearing oil. The oil removes the alcohol, 
renders the section more transparent, and is a solvent for the mount- 
ing media. 

(1) Oil of Cloves. 

Clears quickly, does not spread over the slide, and renders the 
sections somewhat brittle. Dissolves celloidin. Clears from 95^ 
alcohol. 

(2) Oil of Bergamot. 

Clears quickly from 95% alcohol. Does not destroy aniline 
stains. Does not dissolve celloidin. 

(3) Oil of Origanum. 

Does not dissolve celloidin if good. Clears from 95% alcohol. 

(4) Carbolic Acid. 

Clears quickly and does not dissolve celloidin. 

(5) Eycleshymer's Mixture. 

Oil of cedar, one part. 
Oil of bergamot, one part. 
Carbolic acid, one part. 
Does not dissolve celloidin, clears rapidly from 95% alcohol, 
and is a splendid clearing agent. 

(6) Eycleshymer's Nerve Mixture. 

Oil of cedar, 50 c.c. 
Oil bergamot, 50 c.c. 
Carbolic acid, 25 c.c. 

(7) Xylol-Carbolic Acid Mixture. 

Xylol, three parts. 

Carbolic acid crystals, one part. 

(8) Xylol. 

Clears readily after chloroform or absolute alcohol, but not 
after 95% alcohol. Used on paraffin sections only. 

(9) Ether-Alcohol. 

Take equal parts of ether and of absolute alcohol. 



LofC. 



INDEX 



Page 

Acid-f uchsin 84 

Adenoid Tissue 31 

Albumin Fixative, Mayer's 86 

Alcohol, Acid 87 

Alcohol, Ranvier's one-third 84 

Alcohol, as a Fixative 86 

Alimentary Canal 37 

Ammonia Alum 87 

Amoebae 11 

Aorta 29 

Appendix 42 

Areolar Tissue . ._ _ 18 

Bichloride of Mercury 85 

Bladder 49 

Bladder, Epithelium of 14 

Blood 25, 26 

Blood Clot 28 

Blood of Bird 27 

Blood of Frog 27 

BloodVessels 29 

Bone 21 

Borax-carmine 83 

Cartilage 19 

Cartilage , Blastic-fibro 20 

Cartilage, Hyaline 19 

Cartilage, White-fibro 20 

Cartilage, Ossification of 20 

Capillaries, Blood 30 

Capillaries, Bile 45 

Carbolic Acid 88 

Cardiac Glands 38 

Carmine-gelatin 87 

Carnoy's Fluid 86 

Caustic Potash 85 

Cerebellum 79 

Cerebrum 80 

Cerebrum, Fibers of 81 

Celloidin . 87 

Choroid '_ __. 67 

Chrom-acetic Fluid 86 

Chromic Acid 85 



• Page 

Circum vallate Papillae 36 

Clearing Fluids.. 88 

Cochlea ... 69 

Colon _* 41 

Congo Red 83 

Connective Tissues 17 

Connective Tissue, Areolar 18 

Connective Tissue, Embryonic 18 

Connective tissue, White Fibrous. 17 

Cornea 66 

Corpus IyUteum 55 

Corrosive Sublimate 85 

Corti, Organ of 69 

Cowper's Gland 53 

Crystalline Lens, 66 68 

Deiter, Cells of 70 

Duodenum 40 

Ear 69 

Ear, Fcetal 76 

Ehrlich-Biondi Mixture 84 

Elastic Tissue 17 

Elastic Tissue of 1/ung 6f> 

Elastic Tissue Stain — Harris 84 

Elastic Tissue of Trachea 62 

Endothelium 16 

Eosin ._ 84 

Epithelium 12 

Epithelium, Ciliated 14 

Epithelium, Columnar 15 

Epithelium, Squamous Cells.. 12 

Epithelium, Squamous of Frog 13 

Epithelium , Stratified 13 

Epithelium, Transitional 14 

Ether Alcohol 88 

Eycleshymer's Mixture 88 

Eye *_„ 66 

Eyelid 69 

Farrant ' s Gum Glycerin 87 

Fat Cells—.' ,. 19 

Fish, Modified Golgi Method 72 

Fixing Fluids 85 



INDEX— CONTINUED 



Page 

Fixing Fluid, Alcohol 86 

Fixing Fluid, Bichloride of Mer- 
cury 85 

Fixing Fluid, Carnoy's 86 

Fixing Fluid, Chrom-acetic 86 

Fixing Fluid, Flemming's 86 

Fixing Fluid, Miiller's 85 

Fungiform Papillae: 36 

Ganglia 75 

Glycogen 20 

Hsematoxylin Solutions. 82 

Hematoxylin, Bcehrner's 82 

Hematoxylin. Delafield's 82 

Hematoxylin. Ehrlich's Acid 83 

Hematoxylin , Weigert's 83 

Hemin Crystals 28 

Hemin Crystals of Cat, Dog, and 

Rat 28 

Hemin Crystals of a Sparrow 28 

Hemin Crystals from Blood-stain- 
ed Cloth 28 

Harris, Stain of 84 

Hassal, Corpuscles of 32 

Heidenhain , Stain of 84 

Hensen, Cells of 70 

Hydrochloric Acid '.. 85 

Ileum 40 

Imbedding 7 

Imbedding, Celloidin 7 

Imbedding, Paraffin 8 

Intestine 40 

Intestine, Epithelium of 15 

Intestine of Necturus 15 

Karyokinesis 10 

Kidney 46 

Kidney, Macerated 46 

Kidney, Injected 46 

Kidney of Foetal Pig 48 

Larynx 61 

Liver 44 

Liver, Impregnated 45 

Liver, Injected and Stained 44 

Liver of a Pig 45 

Lugol's Solution _ 87 

Lung 62 

Lymphatic Tissue 31 

Lymph Gland : 32 



Page 

Lymph Gland, Cells of 33 

Mammary Gland 57 

Marrow 29 

Mayer's Fixative 86 

Mesothelium 16 

Mitosis . 10 

Miiller, Fluid of 85 

Muscle 23 

Muscle, Cardiac 25 

Muscle, Smooth 25 

Muscle, Striated 23, 24 

Nail 60 

Necturus 15 

Nerve Cells 71 

Nerve Endings .__ 



76 

Nerve Fibers 73, 74 

Nerve Fibers, Medullated __ 74 

Nerve Fibers, Noh-medullated 75 

Nitric Acid 85 

Oil of Bergamot ... 88 

Oil of Cloves 88 

Oil of Origanum 88 

Olfactory Mucous Membrane 70 

Oppel, Method of 45 

Orcein 83 

Ovary 53 

Pancreas 43 

Parotid 42 

Penis 52 

Prostate 53 

Pyloric Stomach 39 

Ranvier's One-third Alcohol 84 

Ranvier's Crosses 77 

Ranvier's Lemon-juice Method 76 

Ranvier's Nodes 74 

Reagents 82 

Reproductive Organs of the Female 53 

Reproductive Organs of the Male _ _ 50 

Retina 68 

Safranin 83 

Scalp 58 

Scalp of a Foetus 59 

Sclera 67 

Skin 57 

Solitary Gland 32 

Spinal Bulb 79 

Spinal Cord 77 



INDEX- Continued 



Page 

Spleen 33 

Spleen , Corpuscles of 35 

Stains 82 

Stain, Acid-fuchsin 84 

Stain, Bcehmer's Hematoxylin __ 82 

Stain, Borax-carmine 83 

Stain, Congo Red 83 

Stain, Delafield's Hematoxylin. _ 82 
Stain, Ehrlich's Acid-hematoxy- 

lin 82 

Stain , Ehrlich-Biondi 84 

Stain, Elastic of Harris 84 

Stain, Eosin 84 

Stain, Iron Hematoxylin 84 

Stain, Orcein 83 

Stain, Safranin 83 

Stain, Thionin_ .. 83 

Stain, Van Gieson's 84 

Stain, Weigert's hematoxylin 83 

Stomach 37 

Stomach, Cardiac glands 38 

Stomach, Injected 38 

Stomach, Pyloric 39 



Page 

Submaxillary Gland 43 

Teeth 22 

Teichmann ' s Crystals 28 

Testis 50 

Thionin 83 

Thymus Gland 32 

Tongue 35 

Tongue, Circum vallate papille of 36 

Tongue, Fungiform Papille of 36 

Tongue , Taste-buds of 36 

Trachea 62 

Thyroid Gland 65 

Ulnar Nerve 74 

Ureter 50 

Uterus 56 

Vagina 57 

Van Gieson's Stain 84 

Vein l__ 30 

Vermiform Appendix 42 

Washing of Tissues __ 6 

Weigert' s Hematoxylin 83 

Xylol 88 

Xylol-carbolic Acid Mixture 88 



HO* 2B '. 



